Compositions and methods of nematode control

ABSTRACT

The present invention provides compositions and methods for identifying agents that stimulate or inhibit nematode reproduction, especially related to oocyte maturation, sheath cell contraction, and ovulation. It is disclosed that the major sperm protein (MSP) is acts in signal transduction of female sexual maturation in nematodes. Provided are compositions and methods for identifying anti-nematode agents with MSP as a target and for controlling nematode populations. MSP is an excellent target for identification of anti-nematode factors because it is highly conserved among members of the phylum Nematoda and is not known to exist in other organisms, especially crops, livestock, pets, and humans. Thus, anti-nematode agents that target MSP are less likely to induce severe side effects when administered to a host and the nematode is unlikely to develop resistance to a highly conserved molecule involved in sexual reproduction.

FILED UNDER 35 U.S.C. § 119 WITH RIGHT TO PRIORITY

[0001] This application claims benefit of U.S. patent application Ser.No. 60/205,829 filed on May 19, 2000, entitled “Control of Nematodes,Stimulation of Nematode Resistance, and Screening Methods forIdentifying Anti-Nematode Factors”, incorporated herein by reference inits entirety and U.S. patent application Ser. No. 60/274,358 filed onMar. 08, 2001, entitled “Control of Nematodes”, incorporated herein byreference in its entirety.

[0002] Be it known that we, David Greenstein, a citizen of The UnitedStates of America, residing at 104 Groome Drive, Nashville, Tenn. 37205and Michael A. Miller, a citizen of The United States of America,residing at 2511 Barton Ave., Nashville, Tenn. 37212; have invented newand useful “Compositions and Methods of Nematode Control”.

FIELD OF THE INVENTION

[0003] The present invention is related to compositions and methods ofnematode control and, in particular, to compositions and methods forcontrol of nematode fertility, for identifying anti-nematode agents, andpotentiating host resistance.

BACKGROUND OF THE INVENTION

[0004] Parasitic nematodes infection of plants and animals arewidespread with approximately 3 billion people being infected worldwide,100 million lives lost, and an estimated $80 billion worth of crops lostannually to these organisms. Human conditions include river fever andelephantiasis each of which cause terrible human suffering. Parasiticnematodes are also a major problem in livestock, horses, and pets. Freeliving nematodes also damage plants during feeding, compete for oxygen,and transmit disease. Certain anti-nematode agents are commerciallyavailable. Disadvantages to these agents, such as the carbamates,include their extreme toxicity to most animals and all humankind. Otheragents, such as ivermectin and its derivatives have undesirable toxiceffects and potentially severe side effects especially those related tobehavior and mental health. In addition, ivermectin resistant strains ofnematodes develop relatively quickly and are reported in crops,livestock, and humans.

[0005] It is clear from the widespread and sever nature of the nematodeproblem and the adverse or toxic nature of many nematode treatingagents, that more effective compounds and methods for controllingnematodes and identifying anti-nematode agents are needed.

STATUS OF THE PRIOR ART

[0006] McCarter et al, (1999) discloses that in the absence of sperm,the production of oocytes remains arrested in nematodes.

[0007] Klass, M. R., et al. (1981) discloses that major sperm protein isa structural protein in sperm cells of nematodes.

[0008] It is disclosed that all cells of Caenorhabditis elegans aredirectly observable in the intact Caenorhabditis elegans animal asreviewed in Hubbard and Greenstein (2000).

[0009] Video microscopy is disclosed to be used for observing the latestages of oocyte development (Ward and Carrel, 1979; Albertson, 1984;Albertson and Thomson, 1993; McCarter et al., 1997; Rose et al., 1997;Hall et al., 1999).

SUMMARY OF THE INVENTION

[0010] The present invention provides, in part, compositions and methodsfor controlling nematode populations, identifying anti-nematode agents,enhancing host resistance to nematode infection, and treating plants andanimals for nematodes.

[0011] Although the present invention is not bound by mechanism ortheory, it is related, in part, to the surprising discovery made by theinventors that the major sperm protein (MSP) of nematodes is a molecularsignaling factor which stimulates maturation of the female reproductivesystem in nematodes. Biological activities of MSP in this regardinclude, but are not limited to: oocyte maturation, gonadal sheath cellcontraction, and ovulation.

[0012] One advantage of the present invention is that inhibitors of theMSP signaling mechanism described herein are contemplated to be highlyspecific to inhibiting nematode proliferation and spread while beingnon-toxic to vertebrates because the MSP gene and polypeptide are highlyconserved between groups and divisions within the Phylum Nematoda,including the genera and species. Vertebrates and other non-nematodeorganisms, on the other hand, are not known to have an MSP gene, orprotein. Thus, inhibitors of MSP stimulated female sexual maturation(FSM) are expected to be specific to organisms of the Phylum Nematoda.

[0013] Another advantage of the present invention is that the highsequence conservation observed among MSP from various nematodes suggeststhat resistance to anti-nematode agents that target MSP is likely to beminimal as mutational changes in MSP in are likely to result in areduction of reproductive capacity.

[0014] Still another advantage of the present invention is that, ingeneral, MSP and FSM effective domains thereof, can be prepared insoluble form. Thus, MSP provides an easily handled target for methods ofthe present invention including in high-throughput assays foridentification of MSP binding and FSM blocking agents.

[0015] One aspect of the present invention includes a method foridentifying an anti-nematode agent by contacting a test compound to anematode and monitoring the FSM response, wherein inhibiting testcompounds are selected as anti-nematode agents.

[0016] Another aspect of the present invention includes identifyinganti-nematode agents by selection of major sperm protein binding agentsas many of these agents will also inhibit FSM. Screening methods aredescribed to determine which MSP binding agents also inhibit FSM.

[0017] In a further aspect of the present invention, a reduction or anabsence of MSP signal transduction impairs or virtually eliminatesnematode fertility.

[0018] Still further aspects of the present invention provide methods ofcontrolling nematode populations including, but not limited to: freeliving nematode populations and parasitic nematode populations which, inturn include animal and plant parasitic nematodes.

[0019] Additional aspects, embodiments, and elements of the presentinvention are described below, including in the detailed description ofthe invention, the examples, and the claims. Aspects, embodiments, andelements described herein are not meant to limit the present inventionin any way including to any particular set thereof. Further aspects,embodiments, elements and equivalents thereof, will be readily apparentbased upon the present disclosure and are considered to be within thespirit and scope of the present invention.

BRIEF DESCRIPTION OF THE DRAWINGS

[0020]FIG. 1 shows a cross-section of a nematode and includes adepiction of the reproductive anatomy. Microinjections as describedherein are preferred to be carried out at the point indicated.

[0021]FIG. 2 shows a polyacrylamide gel electrophoresis of crudesperm-conditioned medium (SCM) (left lane) and isolated 14 kDa femalesexual maturation (FSM) stimulating factor (single band in right lane)identified as major sperm protein (MSP) of C. elegans.

[0022]FIG. 3 shows HPLC traces of fractionation of SCM on C-4 and C-18columns. The “+” sign indicates the respective fractions with FSMpositive biological activity.

[0023]FIG. 4 shows mass spectra of the FSM positive fractions from theHPLC purification. The mass spectra confirm MSP-3 and MSP-142 of C.elegans are present in the biologically active SCM. No other factorswere observed or identified in these mass spectra.

[0024]FIG. 5 is a diagram of MSP mediated cellular communication betweenfemale reproductive cell (for example, the oocytes) and sperm.

[0025]FIG. 6 displays an alignment of twenty-seven MSP polypeptides fromC. elegans. The SEQ ID Numbers are not meant to include the N-terminalmost methionine which is believed to be cleaved during processing.

[0026]FIG. 7 displays and alignment of Ascaris suum alpha and beta MSPisoforms and MSP-142 of C-elegans. Again, the SEQ ID Numbers are notmeant to include to N-terminal most methionine which is believed to becleaved during processing.

[0027]FIG. 8 is a bar graph demonstrating bioactive properties of MSP-77and MSP-38, and sperm protein (versus and buffer control) in stimulatingFSM in nematodes. The top panel displays maturations per hour which is abiological measure of oocyte maturation. The center panel displayscontractions per minute which is a biological measure of sheath cellcontraction. The bottom panel displays average displacement (in microns)which is another measure of sheath cell contraction. Measurements aremade for buffer and the shown concentrations of MSP-77, MSP-38, andsperm MSP. The 6His marking denotes that the MSP includes a histidinetag (Qiagen) and was purified using this system.

[0028]FIG. 9 shows an alignment (performed by visual inspection) of theC-termini of MSPs from wide ranging nematode species and demonstratesthat there is a remarkable sequence conservation. The residues numbersrelative to the full length (minus the methionine) polypeptide are106-126 for each of the following. Ascaris suum (As) MSP isoforms alphaand beta (GenBank accession numbers P27439 and P27440) fragment from106-126 are both represented by SEQ ID NO:13 (identical polypeptides).Onchocerca volvulus (Ov) MSP isoforms 1 and 2 (P13262 and P13263) arealso represented by SEQ ID NO:13. Globodera rostochiensis (Gr, thepotato cyst nematode) MSP isoforms 1, 2, and 3 are each represented bySEQ ID NO:14. C. elegans (Ce) MSP isoforms 142 and 33 (P53017 andP53019) are each represented by SEQ ID NO:15.

[0029]FIG. 10 is a bar graph showing that the N-terminal region of MSPis necessary and sufficient for stimulation of oocyte maturation and theC-terminal region is necessary and sufficient for stimulation of sheathcell contraction. The N-terminal residues 1-106 (SEQ ID NO: 16) ofMSP-77 (SEQ ID NO:9) are necessary and sufficient for stimulation ofoocyte maturation (top panel). The C-terminal residues 106-126 (SEQ IDNO:17) of MSP-77 (SEQ ID NO:9) are necessary and sufficient forstimulation of sheath cell contraction (middle panel) and displacement(bottom panel).

DETAILED DESCRIPTION OF THE INVENTION

[0030] Given the human suffering and economic loss due to nematodes, itis critical that effective and safe anti-nematode compounds and methodsfor controlling nematodes are identified. Although not bound bymechanism or theory, the present invention takes advantage of thediscovery by the inventors that nematode major sperm protein regulatesthe fertility of nematodes. Provided herein are compositions and methodsfor inhibiting or blocking major sperm protein action in fertility.

[0031] Certain utilities of the compositions and methods of the presentinvention include, but are not limited to: identifying anti-nematodeagents, manufacturing anti-nematode agents, providing reagents forscreening test compounds for anti-nematode activity, controllingnematode populations, treating animals and plants for nematode infectionor infestation, treating animals and plants for certain negative effectsof nematodes (including environmental or other effects of free livingnematodes), raising host resistance to nematode infection, andprophylactic treatments to retard nematode infection or the spread ofnematodes.

[0032] 1.0 Definitions

[0033] Unless otherwise defined, all technical and scientific terms usedherein have the same meaning as commonly understood by one of ordinaryskill in the art to which this invention pertains. In case of conflict,the present document, including definitions, will control.

[0034] Descriptions of preferred methods and compositions are providedherein, but should not be construed to be limiting.

[0035] As used herein “prophylaxis” or “prophylactic treatment” refersto measures designed to preserve health or retard the spread of disease.“Prophylaxis” or “prophylactic treatment” do not mean herein a certaintyof the preservation of health or a certainty of a halt to a spread of adisease.

[0036] MSP is an abbreviation for major sperm protein.

[0037] FSM is an abbreviation for female sexual maturation.

[0038] FSM is meant to include, but is not necessarily limited to oocytematuration, sheath cell contraction, and ovulation.

[0039] SCM is an abbreviation for sperm-conditioned media.

[0040]C. elegans is an abbreviation for the nematode genus and speciesCaenorhabditis elegans.

[0041]A. suum is an abbreviation for the nematode genus and speciesAscaris suum.

[0042] The term “biological activity” is meant to include, but is notlimited to: FSM, oocyte maturation, sheath cell contraction, andovulation.

[0043] The term “polypeptide” is known in the art, meanings of which areincluded herein. However, in the event of conflict, the term“polypeptide” means herein, an amino acid polymer of two units orgreater (e.g., a dipeptide or greater).

[0044] The term “polynucleotide” is known in the art, meanings of whichare included herein. However, in the event of conflict, the term“polynucleotide” means a nucleic acid polymer of two units or greater(e.g., a dinucleotide or greater).

[0045] The term “isolated polypeptide” refers to a polypeptide that isat least partially removed from the milieu of molecules in which itoccurs in nature.

[0046] The term “isolated polynucleotide” refers to a polynucleotidethat is at least partially removed from the milieu of molecules in whichit occurs in nature. As used herein, “isolated polynucleotide” alsomeans that the polynucleotide is not identical in structure to anaturally occurring genome or fragment of a genome that spans more thanthree distinct, non-overlapping, genomically consecutive genes inlength.

[0047] Additional definitions of specific terms and phrases are providedherein as needed.

[0048] 2.0 Caenorhabditis Elegans as a Model System

[0049]Caenorhabditis elegans, or C. elegans, is a widely acceptedgenetic model system for studying the genes and gene functions of higherorganisms. C. elegans is also a widely accepted model system forstudying all features of other members of the phylum Nematoda. Thesefeatures include germline development, female sexual maturation, oocytematuration, sheath cell contraction, and ovulation (reviewed by Hubbardand Greenstein, 2000).

[0050]C. elegans is a primitive organism which nonetheless shares manyof the essential biological characteristics that are central problemsof, for example, human biology. The worm is conceived as a single cellwhich undergoes a complex process of development, starting withembryonic cleavage, proceeding through morphogenesis and growth to theadult. It has a nervous system including a rudimentary brain, exhibitsbehaviors, and can “learn”. It produces sperm and eggs, mates andreproduces. After reproduction it gradually ages, loses vigor andfinally dies. Certain genetic features of C. elegans have beenextensively characterized and the genome has been sequenced. All 959somatic cells of its transparent body are visible with a microscope, andits average life span (in the normal state) is a mere 2-3 weeks. Thus,C. elegans provides an ideal compromise between complexity andtractability.

[0051] 3.0 Germline Development in Nematoda

[0052] In general, sexual reproduction of nematodes depends oncoordination between meiotic cell cycle progression, gametogenesis, andfertilization. For example, gamete differentiation is coordinated withmeiotic cell cycle transitions so that fertilization produces a diploidzygote capable of completing embryogenesis and growing into a fertileadult. Normally, nematode oocytes arrest during diplotene/diakinesis ofmeiotic prophase (after the completion of meiosis I and meiosis II)while they grow in size. The release of oocytes from this arrest occursduring meiotic maturation during which the nuclear envelope (i.e.,germinal vesicle) breaks down, the cytoskeleton rearranges, and theoocyte prepares for fertilization.

[0053] Typically, the progression of germline development in C. elegans,and members of Nematoda in general, is as follows. During embryogenesisa reproducible and largely invariant cell lineage generates two germlineprecursor cells, Z2/Z3, and two somatic gonadal precursor cells, Z1/Z4,that together comprise the gonadal primordium at hatching (Kimble andHirsh, 1979; Sulston et al., 1983). During post-embryonic development,Z1 and Z4 give rise to the entire somatic gonad (in the hermaphrodite,these structures are distal tip cells (DTCs), sheath cells,spermathecae, and uterus) and Z2 and Z3 give rise to the germ line. Inlater larval stages the germ line contains a stem-cell population thatcontributes cells to the meiotic pathway. Although germline nucleireside in a syncytium at these stages, individual germline nuclei andtheir surrounding cytoplasm are typically referred to as a “germ cell”.Development of the soma and germ line in both sexes, hermaphrodites andmales, is coordinated by intercellular signaling. The self-fertilehermaphrodites are essentially modified females that produce sperm for ashort time early in gametogenesis and then produce exclusively oocytesas adults. Males produce only sperm and can mate with hermaphrodites toproduce cross progeny.

[0054] During post-embryonic development germ cells proliferatemitotically forming approximately 1000 nuclei in hermaphrodites and 500in males. The C. elegans adult hermaphrodite gonad consists of twoU-shaped gonad arms (FIG. 1). The two equivalent gonad arms of the adulthermaphrodite gonad have been described at an ultrastructural level(Hirsh et al., 1976; Hall et al., 1999; see FIG. 1). The distal portionof the gonad contains syncytial germline nuclei surrounded by incompletemembranes. The germ cells are connected to a core cytoplasm, also calledthe rachis. The stem-cell population is restricted to the distal-mostpart of the germ line; germ cells enter meiosis as they move proximally.In hermaphrodites, approximately the first 40 germ cells to entermeiotic prophase in each gonad arm differentiate as spermatocytes whichcomplete meiosis to form approximately 160 sperm during the fourthlarval stage of development. Upon progression to the adult stage, thegerm cells differentiate as oocytes. Oocytes are surrounded by theproximal gonadal sheath cells (see FIG. 1).

[0055] The gonadal sheath cells are somatic cells that appear to playseveral roles important for the structure, integrity, and reproductivefunctions of the gonad (McCarter et al., 1997; Rose et al., 1997). Theten thin gonadal sheath cells can be subdivided into five pairs (1-5)with each pair having a distinct position along the proximal-distal axisof each gonad arm (FIG. 1). These elongated myoepithelial cells liebetween germ cells and the gonadal basal lamina (Hirsh et al., 1976;Kimble and Hirsh, 1979; Strome, 1986; Hall et al., 1999). The distalsheath cells (pair 1) have an unusual cellular structure with aflattened soma pressed into the gonad such that the cytoplasm isconcentrated into a series of wedges that insert between the germ cells.Pair 1 distal sheath cells also extend finger-like filopodia betweendistal germ cells. Pair 2 ensheathes the loop region. The proximalsheath cells (pairs 3-5; see FIG. 1) contain thick and thin filamentsand contract to drive ovulation (Strome, 1986; Myers et al., 1996;McCarter et al., 1997; Rose et al., 1997; Hall et al., 1999). Theproximal sheath cells are positioned in an interdigitating pattern formgap junctions with one another, and are closely apposed to oocytes (Hallet al., 1999). On their basal surfaces the proximal sheath cells attachto the gonadal basal lamina via hemi-adherens junctions which also serveto anchor the actin cytoskeleton and the contractile apparatus withinthe sheath cells. At their apical face, the proximal sheath cells oftenform gap junctions with oocytes. Yolk particles synthesized by theintestine (Kimble and Sharrock, 1983) gain access to oocytes forreceptor-mediated endocytosis (Grant and Hirsh, 1999) by first movingthrough the sheath pores (Hall et al., 1999). The most proximal sheathcells, pair 5, directly attach to the spermatheca. The spermatheca (1per gonad arm) is a flexible accordion-like structure connected to thegonad arm distally and to the uterus proximally. The spermatheca expandsgreatly to accommodate oocytes, which are fertilized as they enter fromthe gonad arm during ovulation.

[0056] 4.0 Female Sexual Maturation

[0057] The phrase “female sexual maturation” is defined herein toinclude, but is not limited to: meiotic maturation, completion of themeiotic divisions, oocyte production, oocyte or ovum maturation, and theevents and processes of sheath cell contraction and ovulation. Incertain embodiments, “maturation” generally relates to the process bywhich an oocyte or an ova becomes competent for being fertilized. Forexample, female sexual maturation includes maturation of a femalereproductive cell and maturation of an oocyte. In another example, asused herein, female sexual maturation also includes a contraction of asheath cell which is considered herein to be a female reproductive cell.Thus certain cells of the nematode reproductive system are referred toas female reproductive cells, even though they are not an oocyte or ovumper say.

[0058] 5.0 Meiotic Maturation, Ovulation, and Completion of the MeioticDivisions

[0059] Fully grown oocytes remain in the diakinesis stage of prophase Iprior to undergoing meiotic maturation, ovulation, and fertilization.The nuclear envelope of the most proximal oocyte breaks down about 5minutes prior to ovulation as it enters meiotic M-phase from prophase(Ward and Carrel, 1979; McCarter et al., 1999). During maturation, theoocyte also undergoes a structural change termed cortical rearrangement(McCarter et al., 1999). These changes within the oocyte coincide with areproducible sequence of somatic motor events mediated by the proximalsheath cells and the distal spermatheca resulting in ovulation. Duringovulation the mature oocyte enters the spermatheca and is fertilized.The fertilized oocyte then passes into the uterus where both meioticdivisions are completed and embryogenesis begins (Albertson, 1984;Albertson and Thomson, 1993; McCarter et al., 1999). McCarter et al,(1999) discloses that in the absence of sperm, the production of oocytesremains arrested in nematodes.

[0060] 6.0 Assay to Identify the Stimulator of Female Sexual Maturation

[0061] The present inventors developed an in vivo assay for femalesexual maturation and used the assay to discover that the major spermprotein (MSP) is the particular stimulator of female sexual maturation.The general procedures used are as follows. Large quantities of sperm(>108) are purified using a modification of methods developed by Klassand Hirsh (1981). Synchronized cultures of fog-2(q71), which are 50%male and 50% female, are used to purify adult males (Lewis and Flemming,1995). Mutations in the fog-2 gene block spermatogenesis in XX animals,transforming them into females, but have no effect on XO animals, whichare fertile males (Schedl and Kimble, 1988). Males are separated fromfemales, larvae, and embryos based on size by sieving through NITEXscreens of various pore sizes. The populations of males isolated in thisway are generally >99% pure. To isolate sperm, males are placed betweentwo PLEXIGLASS plates and smashed in a vice grip (The Home Depot, Inc.).Intact sperm are then purified from the carcasses by filtration throughNITEX filters (20 micron pore size) and washed in M9 phosphate buffer(Sulston and Hodgkin, 1988) using several rounds of low speedcentrifugation and resuspension. Sperm-conditioned medium (SCM) areprepared by incubating purified sperm in M9 for different periods oftime (e.g., 1-12 hours) and removing the sperm by centrifugation andfiltration through a 0.2 micron filter. Microscopic analysis suggeststhat the sperm are not lysing during the incubation. Polyacrylamide gelelectrophoresis (PAGE) also indicates that the sperm are not lysingduring the incubation and also reveals that that an approximately 14 kDaprotein is enriched in SCM. Referring to FIG. 2, an single protein bandat approximately 14 kDa is apparent in the second lane. PAGE results forunfractionated SCM are displayed in lane 1.

[0062] Another aspect of the present invention provides an assay toscreen SCM for maturation- and contraction-inducing activities,comprising microinjecting SCM into the uterus of reduced capacity spermproducing female nematodes (virgin fog-2(q71) females are used incertain preferred embodiments). Maturation and sheath cell contractionare monitored by time-lapse video microscopy (see Rose et al. (1997) fora general description of time-lapse video microscopy of nematodes).Dramatic increases in oocyte maturation and sheath cell contractionrates are observed following injection of SCM. By contrast, no activity,or essentially no activity, is observed following injection of femaleextracts, 1-methyladenine, acetylcholine, oxytocin, or M9 buffer. Theabove described embodiment, provides a bioassay for sperm derivedfactors that promote oocyte maturation and sheath cell contraction. Itis disclosed in the present invention that the activity is, at least inpart, soluble and present in the sperm-conditioned media. This suggeststhat the factor is secreted by the sperm. Thus, it is a discovery of thepresent invention that while the sperm are sometime physically blockedby the valve, a soluble (diffusible) factor is secreted from the spermto affect FSM and this factor penetrates the valve.

[0063] Still another aspect of the present invention providescompositions and methods for fractionation of SCM, and other nematodebiological materials, for isolation of the FSM stimulatory factor. Inone example, fractionation is performed using reversed phase highpressure liquid chromatography (HPLC) on Vydak C-4 and C-18 analyticalcolumns. FIG. 3, a fraction marked by a + sign is the only activefraction recovered when the SCM is fractionated on the C-4 and C-18columns, respectively (as determined using the in vivo FSM assaydescribed herein). The biologically active fraction is analyzed usingMALDI mass spectrometry peptide mapping and sequencing, identificationtechniques which are known in the art (FIG. 4). This result is verifiedby producing MSP-38 (GenBank Ac. # CAA93089) and MSP-142 (GenBank Ac. #CAB03037) in bacteria and purifying the respective isoforms using acommercially available 6-His tagging and protein product purificationsystem. The inserts are cloned into the pQE-30 Type IV Kit availablefrom Qiagen. The vector includes the 6-His tagging system and methodsfor this cloning and use of the 6-His tagging system of identificationand purification of expressed polypeptides are well known. The result isalso confirmed using C. elegans MSP-77. Additional tests by MADLI-MSanalysis demonstrate the C. elegans MSP in SCM is MSP-3 and MSP 142(Miller et al., 2001).

[0064] Microinjection of purified recombinant MSP mimics the biologicalresponse (FSM) seen using the active, fraction purified from SCM.Additionally, the recombinantly produced protein yields the same peptidemap as the MSP from the active SCM fraction when cleaved with trypsinand analyzed using mass spectrometry. These results demonstrate that C.elegans sperm secrete, or otherwise release, the major sperm protein,that the MSP signal is at least partially soluble in the extracellularfluid surrounds the sperm and in buffers, and that MSP dramaticallyincreases FSM (including oocyte maturation, sheath cell contraction, andovulation rates). The concept of MSP directed signal transduction of FSMincluding oocyte maturation, sheath cell contraction, and ovulation isdiagrammed in FIG. 5. Referring to FIG. 5, MSP serves as a simplemolecule for communication between the sperm and non-mature or arrestedoocytes and inactive female reproductive cells that sperm is present andready to fertilize the egg.

[0065] Furthermore, the MSP is believed to be necessary and sufficientfor stimulating that communication or signal transduction of FSM. Whileit is contemplated and other factors may form a web-like network ofupstream and/or downstream signal cascade, it is an advantage in certainembodiments herein that the role of MSP is quite uncomplicated. Thus,MSP provides an excellent target for anti-nematode agents. Also, due tothe high sequence conservation between MSP polypeptides in all knownnematodes, it is contemplated that nematodes will not readily evolveresistance to compounds or agents that target MSP.

[0066] 7.0 MSP is Known as a Structural Protein Localized Within theSperm Cell

[0067] MSP is known in the art as a structural protein of the nematodesperm (Klass, M. R., et al., 1981). Thus, it is a surprising discoveryof the present invention that MSP is also a nematode female sexualmaturation factor (stimulator, signal transduction element, etc.).

[0068] It is widely accepted that motility of nematode sperm is notactin based, but rather is dependent upon MSP structure and function.Inside the sperm cell, dimeric MSP assembles at one end of a fibrouspolymer of dimeric MSP and disassembles at the other end in atreadmill-like fashion which enables the sperm to protrude and withdrawpseudopodia related to motility.

[0069] 8.0 Sequence Conservation Among the Many MSP Sequences Described

[0070] There are likely more than sixty copies of the MSP gene in the C.elegans genome and it is believed that most of these MSP genes aretranscribed. Referring to FIG. 6, twenty-seven MSP polypeptide sequencesare provided corresponding to polypeptides transcribed from apparentlydistinct MSP genes or polynucleotide sequences. Certain other nematodesapparently have fewer copies of MSP. For example, A. suum are believedto have two copies of an MSP gene both of which are believed to betranscribed into polypeptides.

[0071] Twenty-seven polypeptide sequences for FIG. 6 are aligned usingDivide-and-Conquer Multiple Sequence Alignment which is currentlyavailable over the world wide web (www) at the URLhttp://bibiserv.techfak.uni-bielefeld.de/dca/. The server is located atthe Practical Computer Science and Bioinformatics research group whichis run by Robert Giegerich. The physical location is: Robert Giegerich,AG Praktische Informatik, Technische Fakulta.t, Universitat Bielefeld,Postfach 10 01 31, D-33501 Bielefeld, Germany. The parameters used areBlosum 62 predefined substitution matrix, free shift activated,approximate cut positions activated, recursion stop size L set to 20,window size W set to 0, and weight intensity lambda set to 0. Thealgorithm and method are disclosed in Stoye (1998).

[0072] Referring to FIG. 6, it is known that the N-terminal mostmethionine (from the ATG translation start site) is cleaved. It isexpected that both forms (with and without the methionine) of MSPpolypeptides are active in FSM; therefore, the methionine was includedin FIG. 6. However, the references to the SEQ ID Numbers provided inFIG. 6 correspond to MSP polypeptide sequences of 126 amino acids andare without each N-terminal most methionine as shown in FIG. 6.

[0073] Again, referring to FIG. 6, the sequences of the numerous C.elegans MSP display a high degree of sequence homology. Very fewsequence variations are observed. Residues that vary from the global(general) consensus within a column (determined visually) are marked inbold letter and underlined in FIG. 6. Because MSP polypeptide sequences,and even those of the most divergent known nematodes (see below), are sohighly conserved; the preferred method for alignment is by visualinspection. For example, two or more MSP polypeptide sequences can beeasily lined up next to one another on a computer screen or as writtenout on a paper and one moved against another until the majority of thebases match. Percent identity between any two sequences is calculated bycounting the number of residues that do not match, dividing by the totalnumber of residues in the total sequence being compared (or the shortestof the sequences being compared if one of the pair is shorter inlength), multiplying by 100, and expressing the resulting value as apercent.

[0074] Using this approach, one of ordinary skill in the art can easilydetermine an alignment of a given MSP isolated from any member of thephylum Nematoda to another MSP, including to a C. elegans MSP, and inpreferred embodiments the MSP specified in SEQ ID NO:2. It is alsopreferred that the alignment selected is the one which produces thehighest sequence identity as described above.

[0075] Thus, for example, the visual inspection method described aboveis used to align the sequences for the two known MSPs from A. suum(alpha and beta) with MSP-142 of C. elegans (see FIG. 7). Again, verylittle sequence variation is observed between MSP polypeptides fromthese nematodes that are separated by hundreds of millions of years ofevolutionary pressure. This indicates that the structure functionrelationship in MSP is tight and sequence variation likely results in areduced fitness of the organism.

[0076] 9.0 Biological Activities of MSP on FSM are Conserved in PhylumNematoda Ascaris suum is believed to be one of the most widely separatednematode species from C. elegans in terms of both evolution (hundreds ofmillions of years post divergence from the common organism) and in termsof distinctness of the MSP sequence including at the polypeptide level.For example, evaluation of the biological activity of isolated MSP alphaand MSP beta from A. suum in the in vivo FSM assay described aboveserves as a model system for demonstrating that MSP sequences in generalstimulate nematode FSM in all or nearly all member of the phylumNematoda.

[0077] MSP, isoforms alpha from A. suum is isolated from A. suumnematodes or the corresponding nucleotide is cloned and expressed inbacteria, for example. The specific sequence used is Accession NumberP27439 in the NCBI database(maqsvppgdintqpsqkivfnapyddkhtyhikitnaggrrigwaikttnmrrlsvdppcgvldpkekvlmavscdtfnaatedlnndritiewtntpdgaakqfrrewfqgdgmvrrknlpieynl)and is set forth in SEQ ID NO: 11, wherein SEQ ID NO:11 does not includethe N-terminal methionine in order to represent the polypeptide that isbelieved to be cleaved during cellular processing (see FIG. 7).

[0078] MSP, isoforms beta from A. suum is isolated from A. suumnematodes or the corresponding nucleotide is cloned and expressed inbacteria, for example. The specific sequence used is Accession NumberP27440(maqsvppgdintqpgskivfnapyddkhtyhikitnaggrrigwaikttnmrrlgvdppcgvldpkesvlmavscdtfnaatedlnndritiewtntpdgaakqfrrewfqgdgmvrrknlpieynl)in the NCBI database and is set forth in SEQ ID NO: 12, wherein SEQ IDNO:12 does not include the N-terminal methionine in order to representthe polypeptide that is believed to be cleaved during cellularprocessing.

[0079] Microinjection of MSP, isoform alpha or MSP, isoform beta intothe sperm defective (or reduced sperm capacity) female C. elegans asdescribed above results in a restoration of apparently normal FSMbiological activity even though no sperm are added. This providessupport for using any combination of MSP polypeptide including asexpressed from MSP polynucleotides. Methods known in the art forexpressing polynucleotides that are preferred herein include, but arenot limited to: expression in bacteria, transient expression innematodes, stable expression in nematodes, and transgenic expression innematodes. Transgenic expression in nematodes includes gonadal specificexpression and ectopic expression, such as in transgenic expression insomatic cells of the nematode.

[0080] 10.0 Experiments Demonstrating that MSP Stimulates FSM

[0081] The biological activities of MSP-77 and MSP 38 are studied usingthe in vivo FSM assays described herein. Referring to FIG. 8, MSPisolated from male nematodes, as well as MSP produced in bacteriastimulate oocyte maturation and sheath cell contraction when introducedinto female nematodes with reduced sperm formation such as fog-2 mutants(MSP-77 and MSP-38, in this figure, are isolated from the bacteria witha 6His tag. This general protein isolation technique is known in theart). Again, referring to FIG. 8, the top panel displays maturations perhour which is a biological measure of oocyte maturation. The centerpanel displays contractions per minute which is a biological measure ofsheath cell contraction. The bottom panel displays average displacement(in microns) which is another measure of sheath cell contraction.Measurements are made for buffer and the shown concentrations of MSP-77,MSP-38, and sperm MSP. The 6His marking denotes that the MSP includes ahistidine tag (Qiagen) and was purified using this system.

[0082] Similar experiments are performed with MSP-3 and MSP-142 (whichare identified herein to be localized in sperm-conditioned medium), andMSP from C. briggsae and C. remanei. Also, experiments are performedwith MSP from the distantly related A. suum. Each experimentdemonstrates that MSP is essentially interchangeable with regard to itsbiological activities in FSM.

[0083] 11.0 The Carboxyl-Terminus of MSP Shows High SequenceConservation

[0084] Residues 105 through 125 of MSPs derived from nine differentgenera of nematodes show a 100% sequence identity in these 19consecutive amino acid residues (see FIG. 9). These nematodes representfree-living (C. elegans), animal parasites (Ascaris and Onchocerca), andplant parasites (Globodera). (Specifying these genera as free-living,animal parasites, or plant parasites is not meant to limit the rangethat Nematodes inhabit the environment. In general many groups ofnematodes have a diverse range)

[0085] 12.0 Certain Domains Within MSP Differentially Stimulate FSMActivities

[0086] Another aspect of the present invention provides that certaindomains within MSP differentially regulate certain FSM activities. Forexample, FIG. 10 demonstrates that residues 1-106 (SEQ ID NO:16 ) ofMSP-77 (SEQ ID NO:9) preferentially stimulates oocyte maturation, whileresidues 106-126 (SEQ ID NO:17) of MSP-77 (SEQ ID NO:9) preferentiallystimulates the rate of sheath cell contraction and displacement.

[0087] Thus, the biological activities of FSM, including oocytematuration and sheath cell contraction, can be separated and the presentinvention discloses that different domains within MSP are capable ofregulating those activities independently.

[0088] 13.0 Description of MSP Sequence Fragments

[0089] Still another aspect of the present invention includescompositions and methods for identifying and using domains within MSPincluding for differential regulation of the biological activities ofFSM. For example, specific sized segments of MSP polypeptide aresystematically screened for the impact of that domain on FSM. Thisprovides a fine resolution map of the MSP polypeptide with regard to FSMfunction that can be exploited to identify and manufacture highlyspecific anti-nematode agents, for example. This is also useful, forexample, to identify FSM related domains of particularly high sequenceconservation among members of Nematoda and/or to avoid areas that mightinclude a short region that is similar to a gene or polypeptide inanother organism, the targeting of which with anti-organism agents isnot desired.

[0090] In certain embodiments antibodies (polyclonal and/or monoclonal)are generated against each fragment for use in labeling, identification,and an assay of the present invention. In preferred embodiments, theantibodies are raised against those segments of MSP that influence FSM(either positively or negatively). In other embodiments, the antibodiesare raised by injection of the MSP, or the FSM active domain of the MSP,into an animal (in certain embodiments, a non-human animal) usingtechniques known to one of skill in the art for producing antibodies. Inother embodiments, the antibodies and preferably monoclonal antibodiesare produced in cell, such as hybridomas or by recombinant techniquesthat are known in the art. In certain embodiments, the antibodies areproduced in humanized form. This can be accomplished in certainembodiments by injection of the immunogenic fragment of MSP into ahuman.

[0091] 14.0 Production of MSP Polypeptide Fragments

[0092] Segments of MSP polypeptide sequences are readily manufactured bychemical synthesis, for example by solid phase polypeptide manufacture.Alternatively, such segments can be cloned, synthesized in aheterologous cell system, and isolated. Chemical synthesis is preferredfor embodiments wherein the polypeptide is a polymer of 50 residues orfewer. Segments are polypeptides that are generally 10 amino acids ormore in length (referring to consecutive amino acids in an MSP sequencein this section entitled Production of MSP Polypeptide Fragments).Although, in certain embodiments useful segments are contemplated thatinclude fewer than 10 consecutive MSP amino acids. It is believed hereinthat all or nearly all MSPs are essentially interchangeable with eachother in regard to FSM activity. Although differences may be identifiedwhen examining the differential regulation of FSM activity.

[0093] Segments do not include the full length MSP polypeptide sequence.For example, the 126 consecutive amino acids of SEQ ID NO:2 is not asegment. Segments of a 126 amino acid MSP include 125 or fewer ofconsecutive amino acids of the MSP. It is preferred that the segmentincludes 120 or fewer, 110 or fewer, 106 or fewer, 105 or fewer, 100 orfewer, 95 or fewer, 90 or fewer, 85 or fewer, 80 or fewer, 75 or fewer,70 or fewer, 65 or fewer, 60 or fewer, 55 or fewer, 50 or fewer, 45 orfewer, 40 or fewer, 30 or fewer, 25 or fewer, 20 or fewer, 19 or fewer,18 or fewer, 17 or fewer, 16 or fewer, 15 or fewer, 14 or fewer, 13 orfewer, 12 or fewer, 11 or fewer, or 10 or fewer consecutive amino acidsof an MSP. An MSP alignment variant means that amino acids may besubstituted in any given MSP or MSP segment to make that positionidentical to the same position in another MSP molecule including fromdiverse groups of Nematoda. The segments may also be MSP alignmentvariant which may be referred to herein as MSP alignment variantsegments. Functionally equivalent sequences and biological functionalequivalents are also generally considered to be within the spirit andscope of the present invention and are described below.

[0094] Ranges of segments are also provided in certain embodiments ofthe present invention. For example, a 10 amino acid segment may beselected from any portion of the MSP polypeptide. An 11 amino acidsegment may be selected from any portion of the MSP polypeptide.Segments of lengths including 9 amino acids (consecutive in an MSP), 10,12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29,30, 31, 32, 33, 34, 35, 36, 37, 38, 40, 41, 42, 43, 44, 45, 46, 47, 48,49, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 106, 110, 115,116, 117, 118, 119, 120, 121, 122, 123, and 124 are contemplated forcertain embodiments.

[0095] These segments are generally screened for FSM activity or used inassays to identify MSP binding agents (e.g., agents that bind tospecific domains) and to identify anti-nematode agents. Although manysegments are described, they can be screened readily for FSM activitygiven the present disclosure and without undue burden. A preferredmethod of screening is to utility high-throughput assays such as inmicrotitre plates or other multiple-sample format to rapidly examine alarge number of segments. Such general assay are known in the art andare provided herein with regard to MSP and the embodiments of thepresent invention.

[0096] 15.0 Description of Certain MSP Sequences

[0097] Numerous examples of MSP sequences (including polynucleotide andpolypeptide) are known in the art and are useful for various embodimentsof the present invention. Also, additional MSP sequences including fromadditional nematodes types and species can be readily determined,without burden, using conventional cloning techniques and the fact thatmany sequences are described. Thus, for example, primers can be designedto pull out additional polynucleotides from a pool, such as a gene orgenomic library, including by PCR amplification. These polynucleotidecan be expressed using standard cloning techniques and the MSP activityof the resulting polypeptide is measurable based upon the assaysdisclosed herein. Certain known and described MSPs are provided by wayof example in Table 1 below. The MSPs are represented by AccessionNumbers corresponding to files in the publicly available databasemaintained by the National Center for Biotechnology Information (theNCBI database). These files include polynucleotide sequences andpolypeptide sequences of MSP. All information within each fileidentified by Accession Number is hereby incorporated herein byreference.

[0098] The NCBI database is available on the world wide web at URL“http://www.ncbi.nlm.nih.gov/” and is physically located at: NationalCenter for Biotechnology Information; National Library of Medicine;Building 38A, Room 8N805; Bethesda, Md. 20894 TABLE 1 Nucleotide ProteinNematode Identifier Accession Numbers Accession Numbers Mansonellaozzardi AJ404225 CAC20724 AJ404224 CAC20723 AJ404223 CAC20722 AJ404222CAC20721 AJ404221 CAC20720 AJ404220 CAC20719 AJ404219 CAC20718 AJ404218CAC20717 AJ404217 CAC20716 AJ404216 CAC20715 AJ404215 CAC20714 AJ404214CAC20713 AJ404213 CAC20712 AJ404212 CAC20711 AJ404211 CAC20710 AJ404210CAC20709 AJ404209 CAC20708 CAC20742 Onchocerca volvulus AJ404208CAC20741 AJ404207 CAC20740 AJ404206 CAC20739 AJ404205 CAC20738 AJ404204B45528 J04663 A45528 J04662 Ascaris suum X94249 A45944 P27439 P27440AAB23264 CAA63933 Ascaris lumbricoides M15680 AAA29375 Globoderarostochiensis L24501 AAA29148 L24500 AAA29147 L24499 AAA29146Pratylenchus penetrans AAB02264 AAB02263 AAB02262 AAB02251 AAB02250AAB02249 Pratylenchus scribneri AAB02242 AAB02241 AAB02240 AAB02239

[0099] 16.0 An Assay for Female Sexual Maturation

[0100] A method is provided of identifying an anti-nematode agent, bycontacting a test compound to a nematode and monitoring a female sexualmaturation of the nematode, wherein inhibition of the female sexualmaturation indicates that the test compound includes the anti-nematodeagent.

[0101] The in vivo bioassay is useful, for example, to identifysperm-related factors that promote oocyte maturation and gonadal sheathcell contraction. The assay is also useful, as another example, foridentifying agents that inhibit nematode female sexual maturation.

[0102] In certain embodiments of the assay, mutant nematodes areutilized which have a reduced capacity for sperm production, or lack thecapacity altogether. Such mutants are disclosed to have either low ratesof oocyte maturation and sheath cell contraction activity or none atall. The present invention provides compositions and methods for usingthese mutants to screen for factors that stimulate or inhibit femalesexual maturation. In similar embodiments, compositions and methods areprovided that for using transgenic nematodes. These embodiments aredescribed in detail below.

[0103] Test compounds include any compound in general. Preferred testcompounds are soluble in aqueous solution and thus conducive to typicalbiological assay conditions. In certain embodiments, test compounds areselected from any chemical in a library, for example, as maintained by apharmaceutical or other company. In certain embodiments, test compounds,include proteins, glycoproteins, polypeptides, glycopeptides, aminoacids, nucleic acids of any variety, including DNA, RNA, peptide nucleicacid (PNA), carbohydrates, fatty acids, lipids, etc. In certainembodiments the test compound includes any biologically active molecule.

[0104] Test compounds are administered to nematodes by any desirablemethod for determining which of the test compounds has an inhibitoryactivity on nematode fertility, female sexual maturation, or otheractivity described herein. For example, the test compound can bemicroinjected, co-injected, incubated, or fed to the nematodes. Theassay measures the ability of specific test compounds to inhibit MSPstimulation of oocyte maturation, gonadal sheath cell contraction, andovulation using optical monitoring. In addition to ovulation, laying orreleasing of oocytes or embryos from the organism can be monitoredoptically and used as an endpoint of test compound activity. The opticalmonitoring can be enhanced using labeling reagents, such as fluorescent,radioactive, or enzymatic labels. These labels can be attached usingstandard chemistry known in the art to the test compound, a sperm, amajor sperm protein, the oocyte, a sheath cell, etc.

[0105] Methods for monitoring the FSM by video microscopy are disclosedherein. Other methods for monitoring the FSM can include by radiolabelassay, fluorescent assay, etc. (Miller et al., Science 2001).

[0106] Inhibition of FSM generally refers to a reduction or terminationin rate of an FSM event, in certain preferred embodiments. In otherembodiments, inhibition means reduction in reproductive success,fecundity, etc. In other preferred embodiments, inhibition results incontrol of a population of nematodes including a free-living, parasitic,terrestrial, or an aquatic nematodes population.

[0107] 17.0 Assay for Identifying Inhibitors of MSP Signaling

[0108] As described herein, an object of the present invention is toprovide assays for screening test compounds to identify anti-nematodeagents. In certain embodiments, the agents will be inhibitors of femalenematode fertility. In certain embodiments, the agents will beinhibitors of an MSP signal transduction. Such agents generallyinterfere with nematode fertility and are useful as agents for controlof nematodes including free-living and animal and plant parasiticnematodes. Certain embodiments of screening assays for inhibitors of MSPsignal transduction follow.

[0109] 18.0 Mutant Nematode Strains

[0110] In general, MSP protein is administered to a mutant hermaphroditeor female nematode strain that does not produce sperm (e.g., mutants infog-1, fog-2, fog-3, fem-1, fem-2, fem-3, or gld-1(Fog). The MSP proteincan be administered, for example, by microinjection into the uterus ofthe mutant hermaphrodite or female nematode strain. Such strains areavailable from the C. elegans Genetic Center or natural isolates ofgonochoristic species. In embodiments that use microinjection, thetechnique is carried out according to standard practice (e.g., seeLaMunyon and Ward, Genetics (1994) 138:689-692, incorporated herein byreference). Female nematode sexual maturation, such as oocytematuration, sheath cell contraction, and ovulation are generallyobserved optically. Certain methods are described by McCarter et al.,supra.

[0111] 19.0 Wild-Type Nematode Strains

[0112] In general, methods for screening test compounds for identifyingfactors that inhibit nematode fertility, and preferably that inhibitfemale sexual maturation, can be administered to wild-type nematodes andthe effect of the test compounds is usually monitored optically.Typically, the test compounds can be co-injected, incubated, or fed tonematodes. Ordinarily the endpoint of the assay measures the ability ofcompounds to inhibit MSP stimulation of oocyte maturation, gonadalsheath cell contraction, and ovulation as described above.

[0113] 20.0 Transgenic Nematode Strains

[0114] Nematode strains that ectopically express MSP (including innon-spermatogenic tissues) can be used in methods for screening testfactors to identify anti-nematode agents. Transgenic nematodesexpressing MSP are generated using standard methods (e.g., Methods inCell Biology, ed. H. Epstein and D. Shakes, San Diego Academic Press,452-482, incorporated by reference). Inhibitor compounds are, forexample, incubated with or fed to the transgenic nematodes. The assaymeasures the ability of compounds to inhibit nematode fertilityincluding, but not limited to: MSP stimulation of oocyte maturation,gonadal sheath cell contraction, and ovulation as described above.Typically, the inhibition of nematode fertility is measured optically.Test factors that inhibit MSP signal transduction will inhibit thenematode female sexual maturation. In addition, most transgenic strainsproduce different amounts of the transgenic factor, as is known in theart. Thus, nematodes that transgenically express varying amounts of MSPcan be utilized to determine anti-nematode agent concentrations that areoptimized for use on a particular nematode given that differentnematodes express varying levels of MSP in the wild.

[0115] 21.0 MSP Binding Assay

[0116] Compounds are screened for MSP binding affinity. Panels ofcandidate molecules are affixed to a matrix, for example microtitrewells, using standard methods. Labeled MSP protein, such asfluorescently, radioactively, or enzymatically linked MSP protein, isincubated with the compounds attached to the matrix, and then washed off(under conditions that remove unbound labeled MSP protein). Compoundswhich bind MSP are recognized by retention of the label (for example,optical recognition). Alternatively, MSP protein is affixed to a matrix,for example microtitre wells, and incubated with labeled test compounds,such as fluorescently, radioactively, or enzymatically linked compounds,and then washed off, (under conditions that remove unbound labeledcompound). Compounds which bind MSP are recognized by retention of thelabel.

[0117] In certain embodiments, compounds which bind MSP are then testedfor the ability to block MSP signaling, for example by using bio-assaysdescribed herein. Compounds that inhibit or block nematode reproductionor fertility are anti-nematode agents. In certain embodiments, theanti-nematode agents are used to treat parasitic nematode infections inplants and animals by administering a therapeutically effective amountof the anti-nematode agent to the plant or animal to inhibit, or incertain cases to virtually block, nematode reproduction in the infectedplant or animal.

[0118] 22.0 Regulation of MSP Protein to Protein Interactions

[0119] Compounds are screened for the ability to regulate protein toprotein interactions of MSP. For example, MSP is known to exist inmonomeric and dimeric forms. Thus, test compounds are screened inbiological assays to identify factors that prevent dimerization,multimerization (complexes with two or more MSP subunits), and forfactors that prevent multimers from dissociating into monomers. Theeffect test compounds on multimer formation can be determined byincubating the test compound with the MSP under multimer associating anddissociating conditions. These samples can be tested for biologicalactivity in regard to nematode fertility as described herein. Multimerand monomer formation and dissociation can be monitored by techniquesknown in the art. For example, SDS versus native gel electrophoresis(polyacrylamide gel electrophoresis), electrospray mass spectroscopy,and gel exclusion.

[0120] In certain embodiments, compounds which regulate multimerizationof MSP (formation and dissociation of multimers/monomers) are thentested for the ability to block MSP signaling. For example by usingbiological assays measuring nematode female sexual maturation asdescribed herein. Compounds that inhibit nematode reproduction areanti-nematode agents. In certain embodiments, the anti-nematode agentsare used to treat parasitic nematode infections in plants and animals byadministering a therapeutically effective amount of the anti-nematodeagent to the plant or animal to inhibit, or in certain cases tovirtually block, nematode reproduction in the infected plant or animal.

[0121] 23.0 Certain Nematode Varieties

[0122] Nematoda includes the roundworms and threadworms, and comprises aphylum of generally smooth-skinned, unsegmented worms with a longcylindrical body shape tapered at the ends; the phylum includesfree-living and parasitic forms both aquatic and terrestrial (adaptedfrom Academic Press Dictionary of Science and Technology).

[0123] Table 2, below, provides a listing of the common name andscientific name of a multitude of nematode varieties. MSP genes andproteins can be derived from these or other nematode varieties andstrains for use in conjunction with the present invention (e.g., in ascreening assay). Also, parasitic nematode infections of these or othertypes of nematodes may be treated by anti-nematode agents describedherein or identified as described herein. This list is not meant to belimited on the scope of the invention, but merely to be exemplary oftypes of nematode. Animal parasitic nematodes are also described insupplementary materials appended to this provisional application. TABLE2 Common Name Nematode Genus and species African spiral nematodeHelicotylenchus africanus Alfalfa root nematode Heterodera goettingianaAlmond cyst nematode Heterodera amygdali Amaranth cyst nematodeCactodera amaranthi American dagger nematode Xiphinema americanumAmu-Darya nematode Heterodera oxiana Apple cyst nematode Globodera maliApple root-knot nematode Meloidogyne mali Awl nematodes Dolichodorusspp. Banana meadow nematode Pratylenchus coffeae Banana nematodePratylenchus musicola Banana root-lesion nematode Pratylenchus coffeaeBanana spiral nematode Helicotylenchus multicinctus Banana-root nematodeRadopholus similis Barley cyst nematode Heterodera hordecalis Barleyroot-knot nematode Meloidogyne nassi Beachgrass root-knot nematodeMeloidogyne sasseri Beer nematode Panagrellus silusiae Beet nematodeHeterodera schachtii Beet stem nematode Ditylenchus dipsaci Begonia leafnematode Aphelenchoides fragariae Bentgrass nematode Anguina agrostisBermudagrass cyst nematode Heterodera cardiolata Birch cyst nematodeCactodera betulae Black currant nematode Aphelenchoides ritzemabosiBlueberry root-knot nematode Meloidogyne carolinensis Boxwood spiralnematode Rotylenchus buxophilus Brassica root eelworm Heteroderacruciferae Brassica root nematode Heterodera cruciferae Brazilianroot-knot nematode Meloidogyne exigua Brazilian root-knot nematodeMeloidogyne inornata British root-knot nematode Meloidogyne artielliaBritish spiral nematode Scutellonema brachyurum Buckwheat cyst nematodeHeterodera graduni Bud and leaf nematodes Aphelenchoides spp. Bud andstem nematode Ditylenchus dipsaci Bulb and stem nematodes Ditylenchusspp. Bulb nematode Ditylenchus dipsaci Bulb or stem nematodesDitylenchus spp. Burrowing nematode Radopholus similis Burrowingnematodes Radopholus spp. Cabbage cyst nematode Heterodera cruciferaeCabbage nematode Heterodera cruciferae Cabbage root nematode Heteroderacruciferae Cactus cyst nematode Cactodera cacti Cajanus cyst nematodeHeterodera cajani California dagger nematode Xiphinema index Californiameadow nematode Pratylenchus neglectus California root-lesion nematodePratylenchus neglectus California sessile nematode Cacopaurus epacrisCamel thorn cyst nematode Heterodera oxiana Camellia root-knot nematodeMeloidogyne camelliae Canadian root-knot nematode Meloidogyne microtylaCarnation pin nematode Paratylenchus curvitatus Carnation pin nematodeParatylenchus dianthus Carolina spiral nematode Scutellonema brachyurumCarrot cyst nematode Heterodera carotae Carrot root nematode Heteroderacarotae Cereal cyst nematode Heterodera avenae Cereal cyst nematodeHeterodera latipons Cereal root nematode Heterodera avenae Cerealroot-knot nematode Meloidogyne nassi Cereals root eelworm Heteroderamajor Cereals root nematode Heterodera avenae Chamber's dagger nematodeXiphinema chambersi Christie's spiral nematode Scutellonema christieiChristie's stubby root nematode Trichodorus christiei Chrysanthemumfoliar nematode Aphelenchoides ritzemabosi Chrysanthemum leaf nematodeAphelenchoides ritzemabosi Chrysanthemum nematode Aphelenchoidesritzemabosi Citrus nematode Tylenchulus semipenetrans Citrus ringnematode Criconemoides citri Citrus root nematode Tylenchulussemipenetrans Citrus root-knot nematode Meloidogyne indica Citrus spinenematode Criconema civellae Clover cyst nematode Heterodera trifoliiClover root nematode Heterodera trifolii Clover stem nematodeDitylenchus dipsaci Cobb's awl nematode Dolichodorus heterocephalousCobb's lance nematode Hoplolaimus galeatus Cobb's meadow nematodePratylenchus penetrans Cobb's ring nematode Criconemoides simile Cobb'sroot lesion nematode Pratylenchus penetrans Cobb's root-knot nematodeNacobbus batatiformis Cobb's spiral nematode Helicotylenchusmulticinctus Cobb's stubby root nematode Trichodorus primitivus Coconutnematode Rhadinaphelenchus cocophilus Coconut palm nematodeRhadinaphelenchus cocophilus Cocopalm nematode Rhadinaphelenchuscocophilus Coffee meadow nematode Pratylenchus coffeae Coffee root-knotnematode Meloidogyne exigua Coffee root-lesion nematode Pratylenchuscoffeae Columbia nematode Hoplolaimus columbus Columbia root-knotnematode Meloidogyne chitwoodi Corn cyst nematode Heterodera zeae Cornmeadow nematode Pratylenchus zeae Corn root-lesion nematode Pratylenchuszeae Cotton root-knot nematode Meloidogyne incognita acrita Cowpea cystnematode Heterodera vigni Crown-headed lance nematode Hoplolaimustylenchiformis Currant nematode Aphelenchoides ribes Cyperus cystnematode Heterodera mothi Cyst nematodes Globodera spp. Cyst nematodesHeterodera spp. Cyst-forming nematodes Heterodera spp. Cystoid bodynematodes Meloidoderita spp. Cystoid nematodes Meloidodera spp. Daggernematodes Xiphinema spp. De Man's meadow nematode Pratylenchus pratensisDe Man's root-lesion nematode Pratylenchus pratensis Dock cyst nematodeHeterodera rosii Douglas Fir nematode Nacobbodera chitwoodi Ear-cocklenematode Anguina tritici Estonian cyst nematode Cactodera estonicaEucalypt cystoid nematode Cryphodera eucalypti European dagger nematodeXiphinema diversicaudatum False root-knot nematode of Nacobbusbatatiformis sugar beets False root-knot nematode Nacobbus spp. Fernnematode Aphelenchoides fragariae Fern nematode Aphelenchoides olesistusFescue leaf gall nematode Anguina graminis Ficus cyst nematodeHeterodera fici Fig cyst nematode Heterodera fici Fig pin nematodeParatylenchus hamatus Fig spine nematode Criconema decalineatum Foliarnematodes Aphelenchoides spp. Galeopsis cyst nematode Heteroderagaleopsidis Galeopsis root nematode Heterodera galeopsidis Gall-formingnematodes Meloidogyne spp. Godfrey's meadow nematode Pratylenchusbrachyurus Godfrey's root-lesion nematode Pratylenchus brachyurusGold-plated nematode Globodera rostochiensis Golden nematode Globoderarostochiensis Golden nematode of potato Globodera rostochiensis Grasscyst nematode Punctodera punctata Grass root-gall nematode Subanguinaradicicola Grass sheath nematode Hemicycliophora similis Grass spiralnematode Helicotylenchus erythrinae Great root nematode Heteroderaavenae Hairy-gall nematode Nacobbus batatiformis Heart-shaped cystnematode Heterodera cardiolata Hop cyst nematode Heterodera humuli Hopnematode Heterodera humuli Hop root nematode Heterodera humuliHorsenettle cyst nematode Globodera tabacum virginiae Indian root-knotnematode Meloidogyne brevicauda Iris nematode Ditylenchus destructorJapanese cyst nematode Heterodera elachista Javanese root-knot nematodeMeloidogyne javanica Kansas cyst nematode Heterodera longicollaKidney-shaped nematode Rotylenchulus reniformis Kikuyu grass nematodeMeloidogyne kikuyuensis Knapweed nematode Mesoanguina picridis Knawelcyst nematode Heterodera scleranthii Knotweed cyst nematode Cactoderaweissi Kona coffee root-knot nematode Meloidogyne konaensis Lancenematodes Hoplolaimus spp. Lesion nematodes Pratylenchus spp. Lespedezacyst nematode Heterodera lespedezae Lucerne cyst nematode Heteroderamedicaginis Maple root-knot nematode Meloidogyne ovalis Meadow nematodesPratylenchus spp. Mediterranean cereal cyst nematode Heterodera latiponsMilfoil cyst nematode Globodera millefolii Motha cyst nematodeHeterodera mothi Mushroom nematode Aphelenchoides composticola Mushroomspawn nematode Ditylenchus myceliophagus Needle nematodes Longidorousspp. Nettle cyst nematode Heterodera urticae Nigerian dagger nematodeXiphinema nigeriense Northern root-knot nematode Meloidogyne haplaNutgrass cyst nematode Heterodera cyperi Oak root-knot nematodeMeloidogyne querciana Oak sheathoid nematode Hemicriconemoides biformisOat cyst nematode Heterodera avenae Oat cyst nematode Heterodera majorOat nematode Heterodera avenae Oat root nematode Heterodera avenae Onionstem nematode Ditylenchus dipsaci Osborne's cyst nematode Globoderatabacum solanacearum Pacific dagger nematode Xiphinema radicicola Peacyst nematode Heterodera goettingiana Pea root eelworm Heteroderagoettingiana Pea root nematode Heterodera goettingiana Peanut root-knotnematode Meloidogyne arenaria Persian sessile nematode Cacopaurus pestisPhlox stem nematode Ditylenchus dipsaci Pigeon pea cyst nematodeHeterodera cajani Pin nematodes Paratylenchus spp. Pine cystoid nematodeMeloidodera floridensis Pine sheathoid nematode Hemicriconemoidesfloridensis Pine sting nematode Belonolaimus gracilis Pine wood nematodeBursaphelenchus xylophilus Potato cyst eelworm Globodera rostochiensisPotato cyst nematode Globodera pallida Potato cyst nematode Globoderarostochiensis Potato nematode Globodera rostochiensis Potato rooteelworm Globodera rostochiensis Potato root nematode Globoderarostochiensis Potato rot nematode Ditylenchus destructor Potato tubereelworm Ditylenchus destructor Potato tuber nematode Ditylenchusdestructor Pseudo root-knot nematode Hypsoperine graminis Ramie pinnematode Paratylenchus elachistus Red ring nematode Rhadinaphelenchuscocophilus Reniform nematode Rotylenchulus reniformis Reniform nematodesRotylenchulus spp. Rice blind root nematodes Hirschmanniella spp. Ricecyst nematode Heterodera oryzae Rice nematode Aphelenchoides oryzae Riceroot nematode Hirschmanniella oryzae Rice root-knot nematode Meloidogynegraminicola Rice stem nematode Ditylenchus angustus Rice stunt nematodeTylenchorhynchus martini Rice white-tip nematode Aphelenchoides besseyiRice-root nematode Radopholus oryzae Ring nematodes Criconema spp. Ringnematodes Criconemoides spp. Root nematodes Heterodera spp. Rootnematodes Hirschmanniella spp. Root-gall nematodes Meloidogyne spp.Root-knot nematodes Meloidogyne spp. Root-lesion nematodes Pratylenchusspp. Round cystoid nematode Thecavermiculatus andinus Rubber cystnematode Heterodera fici Rumex cyst nemtode Heterodera rumicisScribner's lesion nematode Pratylenchus scribneri Scribner's meadownematode Pratylenchus scribneri Scribner's root-lesion nematodePratylenchus scribneri Sedge cyst nematode Heterodera cyperi Seed gallnematode Afrina wevelli Seed gall nematodes Anguina spp. Seed-gallnematode Anguina tritici Seinhorst's stubby root nematode Trichodoruspachydermis Sessile nematodes Cacopaurus spp. Seville root-knot nematodeMeloidogyne hispanica Sheath nematodes Hemicycliophora spp. Sheathoidnematodes Hemicriconemoides spp. Shoot gall nematodes Anguina spp.Smooth-headed lesion nematode Pratylenchus brachyurus Smooth-headedmeadow nematode Pratylenchus leiocephalus Smooth-headed nematodePratylenchus brachyurus Sorghum root-knot nematode Meloidogyne acroneaSour paste nematode Panagrellus redivivus South African pin nematodeParatylenchus curvitatus Southern root-knot nematode Meloidogyneincognita Sowthistle cyst nematode Heterodera sonchophila Soybean cystnematode Heterodera glycines Spear nematodes Dorylaimus spp. Spinenematodes Criconema spp. Spiral nematodes Helicotylenchus spp. Spiralnematodes Rotylenchus spp. Spiral nematodes Scutellonema spp. Springcrimp nematode Aphelenchoides fragariae Spring dwarf nematodeAphelenchoides besseyi Steiner's spiral nematode Helicotylenchusdihystera Stem gall nematode Pterotylenchus cecidogenus Stem nematodeDitylenchus dipsaci Sting nematode Belonolaimus gracilis Sting nematodeBelonolaimus longicaudatus Sting nematodes Belonolaimus spp. Strawberrybud nematode Aphelenchoides besseyi Strawberry bud nematodeAphelenchoides fragariae Strawberry foliar nematode Aphelenchoidesfragariae Strawberry nematode Aphelenchoides fragariae Stubby rootnematode Trichodorus christiei Stubby root nematode Trichodoruskurumeensis Stubby root nematodes Paratrichodorus spp. Stubby rootnematodes Trichodorus spp. Stunt nematode Tylenchorhynchus claytoniStunt nematodes Tylenchorhynchus spp. Stylet nematodes Tylenchorhynchusspp. Sugar beet cyst nematode Heterodera schachtii Sugar beet nematodeHeterodera schachtii Sugar cane cyst nematode Heterodera sacchari Sugarcane cyst nematode Heterodera schachtii Sugar cane stylet nematodeTylenchorhynchus martini Summer dwarf nematode Aphelenchoides fragariaeSycamore root-knot nematode Meloidogyne platani Tadzhik cyst nematodeHeterodera tadshikistanica Tadzhik cystoid nematode Meloidoderatadshikistanica Tadzhik root-knot nematode Meloidogyne tadshikistanicaTarjan's sheath nematode Hemicycliophora parvana Tea root-knot nematodeMeloidogyne brevicauda Teasel nematode Ditylenchus dipsaci Tesselatestylet nematode Tylenchorhynchus claytoni Thames' root-knot nematodeMeloidogyne thamesi Thorne's cyst nematode Cactodera thornei Thorne'slance nematode Rotylenchus uniformis Thorne's meadow nematodePratylenchus thornei Thorne's needle nematode Longidorus sylphusThorne's root-lesion nematode Pratylenchus thornei Tobacco cyst nematodeGlobodera tabacum Tobacco stunt nematode Tylenchorhynchus claytoni Tuliproot nematode Ditylenchus dipsaci Turf spiral nematode Rotylenchuschristiei Turkmen cyst nematode Heterodera turcomanica Ustinov cystnematode Heterodera ustinovi Valentine cyst nematode Heteroderacardiolata Vinegar eels Turbatrix aceti Vinegar nematode Turbatrix acetiWalnut meadow nematode Pratylenchus vulnus Walnut root-lesion nematodePratylenchus vulnus Walnut sessile nematode Cacopaurus pestis Wesson'ssheathoid nematode Hemicriconemoides wessoni West African spiralnematode Scutellonema blaberum Wheat cyst nematode Heterodera latiponsWheat gall nematode Anguina tritici White-tip nematode Aphelenchoidesbesseyi Willow cyst nematode Heterodera salixophila Yam nematodeScutellonema bradys Yarrow cyst nematode Globodera achilleae Zimmerman'sspiral nematode Helicotylenchus erythrinae Zoysia spine nematodeCriconema spinalineatum

[0124] 24.0 Treating a Parasitic Nematode Infection

[0125] In certain embodiments of the present invention, a parasiticnematode infection is treated in an infected organism (including plantsand animals). A preferred method of treating a parasitic nematodeinfection is to inhibit nematode fertility or reproduction in theinfected animal. In general, this is done by administering atherapeutically effective amount of an anti-nematode agent as describedherein which disrupts a biological activity of MSP related to femalenematode sexual maturation. The anti-nematode agent can be identified asdescribed in the present invention.

[0126] In certain embodiments, a method of inhibiting a reproduction ofa nematode is provided, comprising inhibiting a signal transduction of amajor sperm protein of the nematode, wherein the signal transductionstimulates a female sexual maturation.

[0127] In certain embodiments, MSP signal transduction is inhibited by amethod comprising administering an MSP-specific antibody (SeveralMSP-specific antibodies have been described in the art. For example,see, Klass, M. R., and Hirsh, D. 1981. Sperm isolation and biochemicalanalysis of the major sperm protein from Caenorhabditis elegans. Dev.Biol. 84, 299-312, incorporated herein by reference). (In certainexamples, the MSP-specific antibody can be administered by solublizingthe antibody in the nematode's environment or by microinjection into theuterus of the nematode. Without being bound to mechanism or theory,these antibodies bind MSP in vivo and inhibit MSP-mediated signaling.Experiments are contemplated wherein hermaphrodite oocyte maturation andsheath cell contraction rates will be compared to hermaphroditesinjected with an antibody which does not bind MSP (a suitable antibodyfor a negative control experiment).

[0128] In certain embodiments, MSP signal transduction is inhibited byadministering a vaccine (especially in the case of treating an animal)wherein the vaccine is developed to an MSP protein or fragment thereof.Any variety of MSP protein, or fragment thereof, can be used in vaccinedevelopment. Many nematode types are described herein and the MSP orMSPs isolated therefrom may be used. Vaccine development protocols arewell known.

[0129] In certain embodiments the identified anti-nematode agent isapplied to crops including by spraying a field to distribute the agent.The agent may gain access directly to the nematode or exist in the soil,water, food or general environment of the nematode. The agent may alsobe transferred to the nematode from a plant or animal.

[0130] 25.0 Pharmaceutical Formulations

[0131] In certain embodiments, the preferred method of administering abiologically active molecule (such as MSP or an anti-nematode agent) isin combination with an excipient (a pharmaceutically acceptablecarrier). The combination of at least one pharmaceutically acceptablecarrier and at least one biologically active molecule is referred toherein as a pharmaceutical formulation.

[0132] The particular excipient is not believed to be critical as longas it is compatible with the biological activity of the biologicallyactive molecule and compatible with administration to the subject,especially plants, animals, human, and other mammals. The choice ofexcipient depends on the nature of the treatment being administered andthe biologically active molecule. The pharmaceutical formulation can beapplied to the surface of the organism being treated for a parasiticnematode infection or injected into the local tissue either in oneapplication or multiple applications. The pharmaceutical formulation canbe combined with additional inert or carrier ingredients and used as atopical salve. The pharmaceutical formulation may also be aerosolized anadministered through the lungs.

[0133] In certain preferred embodiments, a pharmaceutical formulation isprovided along with a method of applying a metered amount of theformulation. For example, if a syringe may include unit markings on thebarrel of the syringe. Typically a syringe will also include a needleand a plunger to form a device effective for administration byinjection. The particular choice of pharmaceutically acceptable carriercan be made by one with skill in the art, such as a treating physician,veterinarian, farmer, or extension service personnel.

[0134] As used herein, “pharmaceutically acceptable carrier” includesany and all solvents, dispersion media, coatings, antibacterial andantifungal agents, isotonic and absorption delaying agents and the likethat are compatible with the biologically active molecule(s) and withadministration to the organism being treated.

[0135] In certain embodiments, the pharmaceutical formulations of thepresent invention are advantageously administered either as liquidsolutions or suspensions. Solid forms may be solubilized or suspended inliquid prior to application or injection. These preparations also may beemulsified. In certain embodiments, a typical composition comprisesabout 50 mg or up to about 100 mg of human serum albumin per milliliterof phosphate buffered saline. Other pharmaceutically acceptable carriersinclude aqueous solutions, salts, preservatives, buffers and the like.Examples of non-aqueous solvents are propylene glycol, polyethyleneglycol, vegetable oil, and organic esters, such as theyloleate. Aqueouscarriers include water, alcoholic/aqueous solutions, and salinesolutions including sodium chloride, Ringer's dextrose, etc.Preservatives include antimicrobial agents, anti-oxidants, chelatingagents and inert gases. The pH and exact concentration of the variouscomponents in the pharmaceutical formulation are adjusted according towell known parameters using well known buffering and dilution agents.

[0136] 26.0 Production of Recombinant MSP Containing Vectors

[0137] Recombinant MSP bacterial strains were produced by cloningMSP-142 and MSP-38 into the pQe-30 6-His vector from Qiagen. Primersspecific for MSP were made that contained a 5′ BamHI site (5′ primer) ora 5′ HindIII (3′ primer) followed by the respective MSP-codingsequences. MSP-38 and MSP-142 were amplified by PCR, cut with BamHI andHindIII, and ligated into the pQe-30 vector (FIG. 6) which was also cutwith BamHI and HindIII. This strategy generated a vector containing anIPTG-inducible promoter followed by an initiator methionine, anN-terminal 6-His tag, and the respective MSP-38 or MSP-142 codingsequences. This construct was then transformed into M15(pREP4) bacterialcells and vector-containing colonies were selected with LB mediumcontaining Ampicillin and Kanamycin. MSP-containing colonies were grownovernight and then MSP expression was induced for 4 hours with 1 mMIPTG. Induced bacteria were pelleted, lysed, and purified using a NiNTAagarose column, which binds the 6-His tag. 6-His purification is knownin the art.

[0138] 27.0 Biological Functional Equivalents of Polynucleotides andPolypeptides

[0139] As is known to one with skill in the art, the biological functionor activity of a gene product may not correspond directly to an absolutepolynucleotide or polypeptide sequence of the gene product. Therefore,the inventor specifically contemplates that alterations to sequencesprovided herein may be made or used wherein the altered sequences, ormethods of use thereof, are equivalent to sequences, or methods of usethereof, and are within the spirit and scope of the present invention.These equivalent sequences are referred to as biologically functionalequivalents, or simply as functional equivalents. Functional equivalentscan include, but are not limited to: conservatively modified variants,degeneracy of the nucleic acid code, polymorphisms, certain insertionsand deletions, and certain length variants. Methods for alteringsequence residues and testing the altered sequences for function oractivity are known in the art or described herein. These alterations maybe natural or made by the “hand of man”.

[0140] At the nucleotide level, different codons can encode the sameamino acid. In other words, the genetic code is degenerate (Alberts etal., Molecular Biology of the Cell, (1989) 2nd Edition, GarlandPublishing, Inc., and incorporated herein by reference). The terms“wobble” and “nucleic acid degeneracy” are used herein to refer tocodons that encode the same amino acid, such as the six codons forarginine or serine. Preferred human codons are provided in materialsappended to this application. Codon preferences for other organisms alsoare well known to those of skill in the art (Wada et al., 1990, supra).Thus, one with skill in the art knows that two different polynucleotidescan encode identical polypeptide sequences due to codon wobble.

[0141] It is understood in the art that amino acid and nucleic acidsequences may include additional residues, such as additional N-terminalor C-terminal amino W acids or 5′ or 3′ sequences, and yet still beessentially as set forth in one of the sequences disclosed herein; solong as the sequence meets the criteria set forth herein, including themaintenance of at least one biological protein activity where proteinexpression is concerned (MSP activity should include at least one typeof stimulation of female nematode sexual maturation). The addition ofterminal sequences particularly applies to nucleic acid sequences thatmay, for example, include various non-coding sequences flanking eitherof the 5′ or 3′ portions of the coding region or may include variousinternal sequences, i.e., introns, which are known to occur within genesbetween coding regions (Alberts et al., supra, incorporated herein byreference). Thus; about 1, 2, 3, 4, 5, 6, 7, or more than 7 amino acidscould be added to a polypeptide and the polypeptide may still retain atleast one biological activity. Or; about 1, 2, 3, 4, 5, 6, 7, or morethan 7 nucleotides could be added to a polynucleotide and expressionproducts of the polynucleotide may still retain at least one biologicalactivity.

[0142] It also is understood in the art that amino acid and nucleic acidresidues may be removed from the N-terminal or C-terminal ends ofpolypeptide or 5′ or 3′ ends of polynucleotide sequences, and yet stillbe essentially as set forth in one of the sequences disclosed herein; solong as the sequence meets the criteria set forth herein, including themaintenance of at least one biological protein activity of where proteinexpression is concerned (in particular stimulating a female nematodesexual maturation with regard to MSP). The removal of terminal sequencesparticularly applies to nucleic acid sequences that may, for example,include various non-coding sequences flanking either of the 5′ or 3′portions of the coding region or may include various internal sequences,i.e., introns, which are known to occur within genes between codingregions (Alberts et al., supra, incorporated herein by reference). Thus;about 1, 2, 3, 4, 5, 6, 7, or more than 7 amino acids could be removedfrom a polypeptide and the polypeptide may still retain at least onebiological activity. Or, about 1, 2, 3, 4, 5, 6, 7, or more than 7nucleotides could be removed from a polynucleotide and expressionproducts of the polynucleotide may still retain at least one biologicalactivity.

[0143] If desired, it is possible using techniques known to one withskill in the art, to include an intron in a recombinant polynucleotidesequence. For example, a bovine growth hormone (bGH) intron includingsplice sites may be added. In certain instances, the addition of anintron to a recombinant polynucleotide has been observed to increaseexpression of the encoded expression product in eukaryotic cells. It isunderstood that the addition of an intron may create a functionallyequivalent sequence.

[0144] It is further understood in the art that insertions and deletionsmay be made within the amino acid and nucleic acid sequence, and yetstill be essentially as set forth in one of the sequences disclosedherein; so long as the sequence meets the criteria set forth herein,including the maintenance of biological protein activity where proteinexpression is concerned (for MSP this should be a stimulation of atleast one female nematode sexual maturation). It is preferred that thereading frame of a polynucleotide sequence be maintained, as is known inthe art (Alberts et al., supra, incorporated herein by reference).

[0145] Excepting intronic or flanking regions, and allowing for thedegeneracy of the genetic code, sequences that have between about 70%and about 79%; or more preferably, between about 80% and about 89%; ormore preferably, between about 90% and about 95% or more; or even morepreferably, between about 96% and about 99%, or more of nucleotidesbeing identical are homologous nucleic acids. Homologous sequences maybe functionally defined as sequences that are capable of hybridizing toa nucleic acid segment under relatively stringent conditions. Suitablerelatively stringent hybridization conditions are well known to those ofskill in the art. In certain embodiments, relatively stringenthybridization conditions allow hybridization between sequences withabout 70% homology or more, but disrupt binding between sequences withless than 70% homology. In certain embodiments, sequences that areconsidered “essentially as set forth” in a sequence listed herein arealso biologically functional equivalents to the listed sequence if atleast one biological activity is found in common.

[0146] At the protein level, peptide sequences that are essentially thesame, in general, are capable of cross-reacting with antibody raisedagainst the respective peptide factor. Methods for isolating, resolving,and analyzing protein/antibody interactions are well known in the artincluding techniques such as SDS-PAGE and Western analysis.

[0147] The nucleic acid segments of the present invention, regardless ofthe length of the coding sequence itself, may be combined with other DNAsequences (one or more of each), such as promoters, polyadenylationsignals, additional restriction enzyme sites, multiple cloning sites,internal ribosome entry sites, introns, other coding segments, membranetransport sequences, and the like, such that their overall length mayvary considerably. It is therefore contemplated that a nucleic acidfragment of almost any length may be employed, with the total lengthpreferably being limited by the ease of preparation and use in theintended recombinant DNA protocol. Therefore, the terms “MSP gene” mayalso comprise any combination of associated control sequences.Furthermore, those skilled in the art of mutagenesis will appreciatethat other analogs, as yet undisclosed or undiscovered, may be used toconstruct MSP analogs (mutants, variants, etc). Additional meaning ofbiological functional equivalents, similarity, percent similarity,equivalents, substantially identical sequences, essentially the same,and essentially similar sequences and activities are described in U.S.Pat. No. 5,922,688 to Hung et al., incorporated herein by reference.

[0148] Naturally, the present invention also encompasses peptides andpolypeptides (or the nucleic acid sequences that encode such peptidesand polypeptides) that contain conservatively modified variants ofsequences of interest, for example, a MSP sequence. One with skill inthe art is able to determine conservative sequence modifications. In thecase of a polypeptide, amino acid substitutions, such as those whichmight be employed in modifying a peptide, such as MSP, are generallybased on the relative similarity of the amino acid side-chainsubstituents, for example, their hydrophobicity, hydrophilicity, charge,size, and the like.

[0149] An analysis of the size, shape and type of the amino acidside-chain substituents reveals that arginine, lysine and histidine areall positively charged residues; that alanine, glycine and serine areall a similar size; and that phenylalanine, tryptophan and tyrosine allhave a generally similar shape. Therefore, based upon theseconsiderations, arginine, lysine and histidine; alanine, glycine andserine; and phenylalanine, tryptophan and tyrosine; are defined hereinas conservative amino acid changes or substitutions. In general,conservatively modified variants of a sequence may include one or moreconservative amino acid change or substitution.

[0150] In making such changes, the hydropathic index of amino acids maybe considered. Each amino acid has been assigned a hydropathic index onthe basis of their hydrophobicity and charge characteristics, these are:isoleucine (+4.5); valine (+4.2); leucine (+3.8); phenylalanine (+2.8);cysteine/cystine (+2.5); methionine (+1.9); alanine (+1.8); glycine(−0.4); threonine (−0.7); serine (−0.8); tryptophan (−0.9); tyrosine(−1.3); proline (−1.6); histidine (−3.2); glutamate (−3.5); glutamine(−3.5); aspartate (−3.5); asparagine (−3.5); lysine (−3.9); and arginine(−4.5).

[0151] The importance of the hydropathic amino acid index in conferringinteractive biological function on a protein is generally understood inthe art (Kyte et al., J. Mol. Biol. (1982) 157(1):105-32, incorporatedherein by reference). It is known that certain amino acids may besubstituted for other amino acids having a similar hydropathic index orscore and still retain a similar biological activity. In making changesbased upon the hydropathic index, the substitution of amino acids whosehydropathic indices are within ±2 is preferred, those which are within±1 are particularly preferred, and those within ±0.5 are even moreparticularly preferred.

[0152] It also is understood in the art that the substitution of likeamino acids can be made effectively on the basis of hydrophilicity. U.S.Pat. No. 4,554,101 to Hopp, incorporated herein by reference, statesthat the greatest local average hydrophilicity of a protein, as governedby the hydrophilicity of its adjacent amino acids, correlates with itsimmunogenicity and antigenicity, i.e. with a biological property of theprotein. It is understood that an amino acid can be substituted foranother having a similar hydrophilicity value and still obtain abiologically equivalent protein.

[0153] As detailed in U.S. Pat. No. 4,554,101, supra, the followinghydrophilicity values have been assigned to amino acid residues:arginine (+3.0); lysine (+3.0); aspartate (+3.0±1); glutamate (+3.0±1);serine (+0.3); asparagine (+0.2); glutamine (+0.2); glycine (0);threonine (−0.4); proline (−0.5±1); alanine (−0.5); histidine (−0.5);cysteine (−1.0); methionine (−1.3); valine (−1.5); leucine (−1.8);isoleucine (−1.8); tyrosine (−2.3); phenylalanine (−2.5); tryptophan(−3.4).

[0154] In making changes based upon similar hydrophilicity values, thesubstitution of amino acids whose hydrophilicity values are within ±2 ispreferred, those which are within ±1 are particularly preferred, andthose within ±0.5 are even more particularly preferred.

[0155] While discussion has focused on conservatively modified variantpolypeptides and functionally equivalent polypeptides arising from aminoacid changes, it will be appreciated that these changes may be effectedby alteration of the encoding polynucleotide; taking into considerationalso that the genetic code is degenerate and that two or more codons maycode for the same amino acid.

[0156] 28.0 Sequence Modification Techniques

[0157] Modifications to sequences, such as MSP sequences, may be madeduring chemical synthesis of the polymers (either nucleotide or peptidesynthesis). It is believed, however, that site-directed mutagenesis ofan encoding nucleic acid, creating a suitably altered polynucleotidesequence is the most cost effective method of generating an alteredpolynucleotide sequence. Where the MSP protein is desired, then themutated sequence may be expressed including in culture (in vitro or exvivo) or in vivo.

[0158] Site-directed mutagenesis is a technique useful in thepreparation of individual peptides, or biologically functionalequivalent proteins or peptides, through specific mutagenesis of theunderlying DNA. Several methods for site directed mutagenesis aredescribed in U.S. Pat. No. 4,873,192 to Kunkel, incorporated herein byreference and in U.S. Pat. No. 4,351,901 to Ball, incorporated herein byreference. The technique further provides a ready ability to prepare andtest sequence variants, for example, incorporating one or more of theforegoing considerations, by introducing one or more nucleotide sequencechanges into the DNA. Site-specific mutagenesis allows the production ofmutants through the use of specific oligonucleotide sequences whichencode the DNA sequence of the desired mutation, as well as a sufficientnumber of adjacent nucleotides, to provide a primer sequence ofsufficient size and sequence complexity to form a stable duplex on bothsides of the deletion junction being traversed. Typically, a primer ofabout 14 to about 25 nucleotides in length is preferred, with about 5 to10 residues on both sides of the junction of the sequence being altered.The primers can be selected by one with ordinary skill in the art basedupon information provided herein, including the Sequence Listings andFigures.

[0159] The technique of site-specific mutagenesis is well known in theart as exemplified by publications (Adelman et al., (1983) DNA2(3)183-193, incorporated herein by reference). As will be appreciated,the technique typically employs a phage vector which exists in both asingle stranded and double stranded form. Typical vectors useful insite-directed mutagenesis include vectors such as the M13 phage. Thesephage are readily commercially available and their use is well known tothose skilled in the art. Double stranded plasmids are also routinelyemployed in site directed mutagenesis which eliminates the step oftransferring the gene of interest from a plasmid to a phage. Kits forphage based site directed mutagenesis are commercially available. Inaddition PCR based methods which may, or may not, involve phage areknown in the art and kits for such purposes are commercially available.

[0160] In certain known techniques, site-directed mutagenesis isperformed by first obtaining a single-stranded vector or melting apartthe two strands of a double stranded vector which includes within itssequence a DNA sequence which encodes the desired nucleotide, such asMSP. An oligonucleotide primer bearing the desired mutated sequence isprepared, generally synthetically as is known to one of ordinary skillin the art. This primer is then annealed with the single-strandedvector, and subjected to DNA polymerizing enzymes such as Escherichiacoli (E. coli) polymerase I Klenow fragment, in order to complete thesynthesis of the mutation-bearing strand. Thus, a heteroduplex is formedwherein one strand encodes the original non-mutated sequence and thesecond strand bears the desired mutation. This heteroduplex vector isthen used to transform appropriate cells, such as E. coli cells, andclones are selected which include recombinant vectors bearing themutated sequence arrangement. Various selection methods that increasethe percentage of specifically modified clones over wild-type are knownand available commercially.

[0161] Kalderon et al. (1984) report several mutagenic methods whichhave proved useful in mutating the native LT gene. Specifically,Kalderon et al. teach deletion mutations by displacement-loopmutagenesis and by the random insertion of Eco RI linkers into the LTgene. Further, point mutation by deletion-loop mutagenesis is taught.The reference also teaches screening procedures for determining thesuccess of such mutations. The teachings of Kalderon et al. (1984)Virology 139(1)109-137 are incorporated herein by reference.

[0162] The preparation of sequence variants of the selected gene usingsite-directed mutagenesis is provided as a method of producingpotentially useful nucleic acids and peptides (such as MSP and MSPvariants) and is not meant to be limiting as there are other ways inwhich sequence variants of these nucleotide and peptides may beobtained. For example, recombinant vectors encoding the desired genesmay be treated with mutagenic agents to obtain sequence variants for themutagenesis of plasmid DNA using hydroxylamine or random mutagenesis maybe performed using the PCR technique.

[0163] Sequence analysis of a potentially mutant nucleic acid sequenceis carried out by methods known in the art, typically by either Sangerdideoxy sequencing (Sanger et al., PNAS (1977) 74:5363-5467,incorporated herein by reference; U.S. Pat. No. 4,871,929 to Barnes; andU.S. Pat. No. 4,962,020 to Tabor et al., each patent incorporated hereinby reference) or automated sequencing (U.S. Pat. No. 5,365,455 toTibbetts et al., incorporated herein by reference).

[0164] In addition to the MSP peptidyl compounds described herein, theinventors also contemplate that other sterically similar compounds maybe formulated to mimic the key portions of the peptide structure. Suchcompounds may be used in the same manner as the peptides of theinvention and hence are also functional equivalents. The generation of astructural functional equivalent may be achieved by the techniques ofmodeling and chemical design known to those of skill in the art. It willbe understood that all such sterically similar constructs fall withinthe scope of the present invention.

[0165] Livestock include, but are not limited to: horses, work horses,show horses, cattle, sheep, goats, and the like. Pets include, but arenot limited to: dogs, cats, horses, birds, and the like.

EXAMPLES Example 1

[0166] Large quantities of sperm (generally >108) are purified using amodification of methods developed by Klass and Hirsh (1981). Adult malesare identified using synchronized cultures (Lewis and Flemming, 1995) offog-2(q71), which are 50% male and 50% female. Mutations in the fog-2gene block spermatogenesis in )XX animals, transforming them intofemales, but have no effect on XO animals, which are fertile males(Schedl and Kimble, 1988). Males are separated from females, larvae, andembryos based on size by sieving through NITEX screens of various poresizes. The populations of males isolated in this way are generally >99%pure. To isolate sperm, males are placed between two PLEXIGLASS platesand smashed in a vice grip (The Home Depot, Inc.). Intact sperm are thenpurified from the carcasses by filtration through NITEX filters (20micron pore size) and washed in M9 phosphate buffer (Sulston andHodgkin, 1988) using several rounds of low speed centrifugation (e.g.,10,000 xg) and resuspension. Sperm-conditioned medium (SCM) is preparedby incubating purified sperm in M9 for different periods of time (1-12h) and subsequently removing the sperm by centrifugation and filtrationthrough a 0.2 micron filter. Microscopic analysis suggests that thesperm are not lysing during the incubation. Polyacrylamide gelelectrophoresis (PAGE) further indicaets that the sperm are not lysingduring the incubation, but that a 14 kDa protein is enriched in SCM(FIG. 2).

REFERENCES

[0167] All references, articles, U.S. patents, Non-U.S. patents,exhibits and the like referred to herein, including those listed belowor attached, are hereby incorporated herein by reference in theirentirety.

[0168] Achanzar, W. E., and Ward, S. (1997). A nematode gene requiredfor sperm vesicle fusion. Journal of Cell Science 110: 1073-1081.

[0169] Albertson, D. G. (1984). Formation of the first cleavage spindlein nematode embryos. Developmental Biology 101: 61-72.

[0170] Albertson, D. G., and Thomson, J. N. (1993). Segregation ofholocentric chromosomes at meiosis in the nematode, Caenorhabditiselegans. Chromosome Research 1: 15-26.

[0171] Anderson, E., and Albertini, D. F. (1976). Gap junctions betweenthe oocyte and companion follicle cells in the mammalian ovary. Journalof Cell Biology 71: 680-686.

[0172] Argon, Y., and Ward, S. (1980). C. elegansfertilization-defective mutants with abnormal sperm. Genetics 96:413-433.

[0173] Aroian, R. V., Field, C., Pruliere, G., Kenyon, C., and Alberts,B. M. (1997). Isolation of actin-associated proteins from Caenorhabditiselegans oocytes and their localization in the early embryo. EMBO Journal16: 1541-1549.

[0174] Aruffo, A., and Seed, B. (1987). Molecular cloning of a CD28 cDNAby a high-efficiency COS cell expression system. Proceedings of theNational Academy of Sciences U S A 84: 8573-8577.

[0175] Austin, J., and Kimble, J. (1987). glp-1 is required in the germline for regulation of the decision between mitosis and meiosis in C.elegans. Cell 51: 589-599.

[0176] Barton, M. K., and Kimble, J. (1990). fog-1, a regulatory generequired for specification of spermatogenesis in the germ line ofCaenorhabditis elegans. Genetics 125: 29-39.

[0177] Bashir, R., Britton, S., Strachan, T., Keers, S., Vafiadaki, E.,Lako, M., Richard, I., Marchand, S,. Bourg, N., Argov, Z., Sadeh, M.,Mahjneh, I., Marconi, G., Passos-Bueno, M. R., Moreira, E. de S., Zatz,M., Beckmann, J. S., and Bushby, K. (1998). A gene related toCaenorhabditis elegans spermatogenesis factor fer-1 is mutated inlimb-girdle muscular dystrophy type 2B. Nature Genetics 20: 37-42.

[0178] Blaxter, M. (1998). Caenorhabditis elegans is a nematode. Science282: 2041-2046.

[0179] Boxem, M., Srinivasan, D. G., and van den Heuvel, S. (1999). TheCaenorhabditis elegans gene ncc-1 encodes a cdc2-related kinase requiredfor M phase in meiotic and mitotic cell divisions, but not for S phase.Development 126: 2227-2239.

[0180] Brenner, S. (1974). The genetics of Caenorhabditis elegans.Genetics 77: 71-94.

[0181] Browning, H., and Strome, S. (1996). A sperm-supplied factorrequired for embryogenesis in C. elegans. Development 122: 391-404.

[0182] Bullock, T. L., Roberts, T. M., and Stewart, M. (1996). 2.5 Aresolution crystal structure of the motile major sperm protein (MSP) ofAscaris suum. Journal of Molecular Biology 263: 284-296.

[0183]C. elegans Sequencing Consortium. (1998). Genome sequence of thenematode Caenorhabditis elegans: A platform for investigating biology.Science 282: 2012-2018.

[0184] Cadigan, K. M., and Nusse, R. (1997). Wnt signaling: a commontheme in animal development. Genes and Development 11: 3286-3305.

[0185] Chase, D., Serafinas, C., Ashcroft, N., Kosinski, M., Longo, D.,Ferris, D. K., and Golden, A. (2000). The polo-like kinase PLK-1 isrequired for nuclear envelope breakdown and the completion of meiosis inCaenorhabditis elegans. Genesis: The Journal of Genetics and Development26: 26-41.

[0186] Chen, P. J., Singal, A., Kimble, J., and Ellis, R. E. (2000). Anovel member of the Tob family of proteins controls sexual fate inCaenorhabditis elegans germ cells. Developmental Biology 217: 77-90.

[0187] Church, D. L., Guan, K.-L., and Lambie, E. J. (1995). Three genesof the MAP kinase cascade, mek-2, mpk-1/sur-1 and let-60 ras, arerequired for meiotic cell cycle progression in Caenorhabditis elegans.Development 121: 2525-2535.

[0188] Clandinin, T. R., DeModena, J. A., and Sternberg, P. W. (1998).Inositol triphosphate mediates a RAS-independent response to LET-23receptor tyrosine kinase activation in C. elegans. Cell 92: 523-533.

[0189] Colledge, W. H., Carlton, M. B. L., Udy, G. B., and Evans, M. J.(1994). Disruption of c-mos causes parthenogenetic development ofunfertilized mouse eggs. Nature 370: 65-68.

[0190] Cordero, M. M., Cornish, T. J., Cotter, R. J., and Lys, I. A.(1995). Sequencing peptides without scanning the reflectron: post-sourcedecay with a curved-field reflectron time-of-flight mass spectrometer.Rapid Communications in Mass Spectrometry 9: 1356-1361.

[0191] Coulson, A., Huynh, C., Kozono, Y., and Shownkeen, R. (1995). Thephysical map of the Caenorhabditis elegans genome. In: Epstein, H. F.,and Shakes, D. C., editors. Methods in Cell Biology. Caenorhabditiselegans: Modern Biological Analysis of an Organism. San Diego: AcademicPress. p. 533-550.

[0192] Cross, D. A., and Smythe, C. (1998). PD98059 preventsestablishment of the spindle assembly checkpoint and inhibits the G2-Mtransition in meiotic but not mitotic cell cycles in Xenopus.Experimental Cell Research 241: 12-22.

[0193] Davis, S., Aldrich, T. H., Valenzuela, D. M., Wong, V. V., Furth,M. E., Squinto, S. P., and Yancopoulos, G. D. (1991). The receptor forciliary neurotrophic factor. Science 253: 59-63.

[0194] Dernburg, A. F., McDonald, K., Moulder, G., Barstead, R.,Dresser, M., and Villeneuve, A. M. (1998). Meiotic recombination in C.elegans initiates by a conserved mechanism and is dispensable forhomologous chromosome synapsis. Cell 94: 387-398.

[0195] Ellis, R. E., and Kimble, J. (1995). The fog-3 gene andregulation of cell fate in the germ line of Caenorhabditis elegans.Genetics 139: 561-577.

[0196] Ferby, I., Blazquez, M., Palmer, A., Eritja, R., and Nebreda, A.R. (1999). A novel p34(cdc2)-binding and activating protein that isnecessary and sufficient to trigger G(2)/M progression in Xenopusoocytes. Genes and Development 13: 2177-2189.

[0197] Ferrell, J. E., Jr. (1999). Xenopus oocyte maturation: newlessons from a good egg. BioEssays 21: 833-842.

[0198] Ferrell, J. E., Jr., Wu, M., Gerhart, J. C., and Martin, G. S.(1991). Cell cycle tyrosine phosphorylation of p34cdc2 and amicrotubule-associated protein kinase homolog in Xenopus oocytes andeggs. Molecular and Cellular Biology 11: 1965-1971.

[0199] Fire, A., Xu, S., Montgomery, M. K., Kostas, S. A., Driver, S. E,and Mello, C. C. (1998). Potent and specific genetic interference bydouble-stranded RNA in Caenorhabditis elegans. Nature 391: 806-811.

[0200] Fisher, D. L., Brassac, T., Galas, S., and Doree, M. (1999).Dissociation of MAP kinase activation and MPF activation inhormone-stimulated maturation of Xenopus oocytes. Development 126:4537-4546.

[0201] Francis, R., Barton, M. K., Kimble, J., and Schedl, T. (1995).gld-1, a tumor suppressor gene required for oocyte development inCaenorhabditis elegans. Genetics 139: 579-606.

[0202] Gavrilets, G., Nature 403, 886 (2000).

[0203] Godeau, J. F., Schorderet-Slatkine, S., Hubert, P., and Baulieu,E. E. (1978). Induction of maturation in Xenopus laevis oocytes by asteroid linked to a polymer. Proceedings of the National Academy ofSciences USA 75: 2353-2357.

[0204] Gotob, Y., Masuyama, N., Dell, K., Shirakabe, K., and Nishida, E.(1995). Initiation of Xenopus oocyte maturation by activation of themitogen-activated protein kinase cascade. Journal of BiologicalChemistry 270: 25898-25904.

[0205] Gotoh, Y., Moriyama, K., Matsuda, S., Okumura, E., Kishimoto, T.,Kawasaki, H., Suzuki, K., Yahara, I., Sakai, H., and Nishida, E. (1991).Xenopus M phase MAP kinase: isolation of its cDNA and activation by MPF.EMBO Journal 10 :2661-2668.

[0206] Grant, B., and Hirsh, D. (1999). Receptor-mediated endocytosis inthe Caenorhabditis elegans oocyte. Molecular Biology of the Cell 10:4311-4326.

[0207] Greenstein, D., Hird, S., Plasterk, R. H. A., Andachi, Y.,Kohara, Y., Wang, B., Finney, M., and Ruvkun, G. (1994). Targetedmutations in the Caenorhabditis elegans POU homeo box gene ceh-18 causedefects in oocyte cell cycle arrest, gonad migration, and epidermaldifferentiation. Genes and Development 8: 1935-1948.

[0208] Greenstein, D., unpublished results.

[0209] Gross, S. D., Schwab, M. S., Taieb, F. E., Lewellyn, A. L., Qian,Y. W., and Mailer, J. L. (2000). The critical role of the MAP kinasepathway in meiosis II in Xenopus oocytes is mediated by p90(Rsk).Current Biology 10: 430-438.

[0210] Haaf, A., Butler, P. J., Kent, H. M., Fearnley, I. M., Roberts,T. M., Neuhaus, D., and Stewart, M. (1996). The motile major spermprotein (MSP) from Ascaris suum is a symmetric dimer in solution.Journal of Molecular Biology 260: 251-260.

[0211] Haccard, O., Lewellyn, A., Hartley, R. S., Erikson, E., andMaller, J. L. (1995). Induction of Xenopus oocyte meiotic maturation byMAP kinase. Developmental Biology 168: 677-682.

[0212] Hall, D. H., Winfrey, V. P., Blaeuer, G., Hoffman, L. H., Furuta,T., Rose, K. L., Hobert, O., and Greenstein, D. (1999). Ultrastructuralfeatures of the adult hermaphrodite gonad of Caenorhabditis elegans:relations between the germ line and soma. Developmental Biology 212:101-123.

[0213] Hashimoto, N., Watanabe, N., Furuta, Y., Tamemoto, H., Sagata,N., Yokoyama, M., Okazaki, K., Nagayoshi, M., Takeda, N., Ikawa, Y., andAizawa, S. (1994). Parthenogenetic activation of oocytes in c-mosdeficient mice. Nature 370: 68-71.

[0214] Hill, D. P., Shakes, D. C., Ward, S., and Strome, S. (1989). Asperm-supplied product essential for initiation of normal embryogenesisin Caenorhabditis elegans is encoded by the paternal-effectembryonic-lethal gene, spe-11. Developmental Biology 136: 154-166.

[0215] Hill, K. L. and L'Hernault, S. W., submitted.

[0216] Hirsh, D., Oppenheim, D., and Klass, M. (1976). Development ofthe reproductive system of Caenorhabditis elegans. Developmental Biology49: 200-219.

[0217] Holst, P. A., and Phemister, R. D. (1971). The prenataldevelopment of the dog: Preimplantation events. Biology of Reproduction5: 194-206.

[0218] Hooper, N. M., and Bashir, A. (1991).Glycosyl-phosphatidylinositol-anchored membrane proteins can bedistinguished from transmembrane polypeptide-anchored proteins bydifferential solubilization and temperature-induced phase separation inTriton X-114. Biochemical Journal 280: 745-751.

[0219] Huang, W., Kessler, D. S., and Erikson, R. L. (1995). Biochemicaland biological analysis of Mek1 phosphorylation site mutants. MolecularBiology of the Cell 6: 237-245.

[0220] Hubbard, E. J. A., and Greenstein, D. (2000). The C. elegansgonad: a test tube for cell and developmental biology. DevelopmentalDynamics 218: 2-22.

[0221] Hyttel, P., Farstad, W., Mondain-Monval, M., Bakke Lajord, K.,and Smith, A. J. (1990). Structural aspects of oocyte maturation in theblue fox (Alopex lagopus). Anatomy and Embryology 181, 325-331.

[0222] Ishikawa, K., Hanaoka, Y., Kondo, Y., and Imai, K. (1977).Primary action of steroid hormone at the surface of amphibian oocyte inthe induction of germinal vesicle breakdown. Molecular and CellularEndocrinology 9: 91-100.

[0223] Italiano, J. E., Jr., Roberts, T. M., Stewart, M., and Fontana,C. A. (1982). Reconstitution in vitro of the motile apparatus from theamoeboid sperm of Ascaris shows that filament assembly and bundling movemembranes. Cell 84: 105-114.

[0224] Jansen, G., Hazendonk, E., Thijssen, K. L., and Plasterk, R. H.(1997). Reverse genetics by chemical mutagenesis in Caenorhabditiselegans. Nature Genetics 17: 119-121.

[0225] Kagiwada, S., Hosaka, K., Murata, M., Nikawa, J., and Takatsuki,A. (1998). The Saccharomyces cerevisiae SCS2 gene product, a homolog ofa synaptobrevin-associated protein, is an integral membrane protein ofthe endoplasmic reticulum and is required for inositol metabolism.Journal of Bacteriology 180: 1700-1708.

[0226] Kanatani, H., Shirai, H., Nakanishi, K., and Kurokawa, T. (1969).Isolation and identification on meiosis inducing substance in starfishAsterias amurensis. Nature 221: 273-274.

[0227] Kaufmann, R., Spengler, B., and Lutzenkirchen, F. (1993). Massspectrometric sequencing of linear peptides by product-ion analysis in areflectron time-of-flight mass spectrometer using matrix-assisted laserdesorption ionization. Rapid Communications in Mass Spectrometry 7:902-910.

[0228] Kimble, J., and Hirsh, D. (1979). The postembryonic cell lineagesof the hermaphrodite and male gonads in Caenorhabditis elegans.Developmental Biology 70: 396-417.

[0229] Kirby, C., Kusch, M., and Kemphues, K. (1990). Mutations in thepar genes of Caenorhabditis elegans affect cytoplasmic reorganizationduring the first cell cycle. Developmental Biology 142: 203-215.

[0230] Klass, M. R., and Hirsh, D. (1981). Sperm isolation andbiochemical analysis of the major sperm protein from C. elegans.Developmental Biology 84: 299-312.

[0231] Kosako, H., Gotoh, Y., and Nishida, E. (1994). Requirement forthe MAP kinase kinase/MAP kinase cascade in Xenopus oocyte maturation.EMBO Journal 3: 2131-2138.

[0232] LaMunyon, C. W., and Ward, S. (1994). Assessing the viability ofmutant and manipulated sperm by artificial insemination ofCaenorhabditis elegans. Genetics 138: 689-692.

[0233] Laurent, F., Labesse, G., and de Wit, P. (2000). Molecularcloning and partial characterization of a plant VAP33 homologue with amajor sperm protein domain. Biochemical and Biophysical ResearchCommunications 270: 286-292.

[0234] Lee, M. -H. and Schedl, T., unpublished results.

[0235] Lewis, J. A., and Flemming, J. T. (1995). Basic culture methods.In: Epstein, H. F., and Shakes, D. C., editors. Methods in Cell Biology.Caenorhabditis elegans: Modern Biological Analysis of an Organism. SanDiego: Academic Press. p. 3-29.

[0236] L'Hernault, S. W. (1997). Spermatogenesis. In: Riddle, D. L.,Blumenthal, T., Meyer, B. J., and Priess, J. R., editors. C. elegans II.Cold Spring Harbor, New York: Cold Spring Harbor Laboratory Press. p.271-294.

[0237] L'Hernault, S. W., and Roberts, T. M. (1995). Cell biology ofnematode sperm. In: Epstein, H. F., and Shakes, D. C., editors. Methodsin Cell Biology. Caenorhabditis elegans: Modern Biological Analysis ofan Organism. San Diego: Academic Press. p. 273-301.

[0238] L'Hernault, S. W., Arduengo, P. M., J. Cell Biol. 119, 55 (1992).

[0239] Lin, R. J., Kao, H. Y., Ordentlich, P., and Evans, R. M. (1998).The transcriptional basis of steroid physiology. Cold Spring HarborSymposium on Quantitative Biology 63: 577-585.

[0240] Liu, X., Kim, C. N., Yang, J., Ronald Jemmerson, R., and Wang, X.(1996). Induction of apoptotic program in cell-free extracts:requirement for dATP and cytochrome c. Cell 86: 147-157.

[0241] MacMorris, M., Spieth, J., Madej, C., Lea, K., and Blumenthal, T.(1994). Analysis of the VPE sequences in the Caenorhabditis elegansvit-2 promoter with extrachromosomal tandem array-containing transgenicstrains. Molecular and Cellular Biology 14: 484-491.

[0242] Masui, Y., and Clarke, H. J. (1979). Oocyte maturation.International Review of Cytology 57: 185-282.

[0243] Masui, Y., and Markert, C. L. (1971). Cytoplasmic control ofnuclear behavior during meiotic maturation of frog oocytes. Journal ofExperimental Zoology 177: 129-145.

[0244] McCarter, J., Bartlett, B., Dang, T., and Schedl, T. (1997).Soma-germ cell interactions in Caenorhabditis elegans: Multiple eventsin germline development require the somatic sheath and spermathecallineages. Developmental Biology 181: 121-143.

[0245] McCarter, J., Bartlett, B., Dang, T., and Schedl, T. (1999). Onthe control of oocyte meiotic maturation and ovulation in C. elegans.Developmental Biology 205: 111-128.

[0246] Mendez, R., Hake, L. E., Andresson, T., Littlepage, L. E.,Ruderman, J. V., and Richter, J. D. (2000). Phosphorylation of CPEbinding factor by Eg2 regulates translation of c-mos mRNA. Nature 404:302-307.

[0247] Miller, M. A., Nguyen V. Q., Lee, M., Kosinski, M., Schedl, M.,Caprioli, R. M., and Greenstein D. (2001) A Sperm Cytoskeletal ProteinThat Signals Oocyte Meiotic Maturation and Ovulation. Science 2001 291:2144-2147.

[0248] Morgan, D. 0. (1997). Cyclin-dependent kinases: engines, clocks,and microprocessors. Annual Review of Cell and Developmental Biology 13:261-291.

[0249] Muhlrad, D., Hunter, R., and Parker, R. (1992). A rapid methodfor localized mutagenesis of yeast genes. Yeast 8: 79-82.

[0250] Mukherjee, S., Ghosh, R. N., and Maxfield, F. R. (1997).Endocytosis. Physiology Reviews 77: 759-803.

[0251] Munson, P. J., and Rodbard, D. (1980). Ligand: a versatilecomputerized approach for characterization of ligand-binding systems.Analytical Biochemistry 107: 220-239.

[0252] Myers, C. D., Goh, P. -Y., Allen, T. St. C., Bucher, E. A., andBogaert, T. (1996). Developmental genetic analysis of Troponin Tmutations in striated and nonstriated muscle cells of Caenorhabditiselegans. Journal of Cell Biology 132: 1061-1077.

[0253] Nance, J., Minniti, A. N., Sadler, C., and Ward, S. (1999).spe-12 encodes a sperm cell surface protein that promotes spermiogenesisin Caenorhabditis elegans. Genetics 152: 209-220.

[0254] Nelson, G. A., Roberts, T. M., and Ward, S. (1982). C. elegansspermatozoan locomotion: Amoeboid movement with almost no actin. Journalof Cell Biology 92: 121-131.

[0255] Nelson, G. A., and Ward, S. (1980). Vesicle fusion, pseudopodextension and amoeboid motility are induced in nematode spermatids bythe ionophore monensin. Cell 19: 457-464.

[0256] Palmer, A. and Nebreda, A. R., Prog. Cell Cycle Res.4, 131(2000).

[0257] Pandey, A., and Mann, M. (2000). Proteomics to study genes andgenomes. Nature 405: 837-846.

[0258] Pavalko, F. M., and Roberts, T. M. (1989). Posttranslationalinsertion of a membrane protein on Caenorhabditis elegans sperm occurswithout de novo protein synthesis. Journal of Cellular Biochemistry 41:57-70.

[0259] Podbilewicz, B. (1996). ADM-1, a protein with metalloprotease-and disintegrin-like domains, is expressed in syncytial organs, sperm,and sheath cells of sensory organs in Caenorhabditis elegans. MolecularBiology of the Cell 7: 1877-1893.

[0260] Posada, J., and Cooper, J. A. (1992). Requirements forphosphorylation of MAP kinase during meiosis in Xenopus oocytes. Science255: 212-215.

[0261] Qian, Y. W., Erikson, E., and Maller, J. L. (1999). Mitoticeffects of a constitutively active mutant of the Xenopus polo-likekinase Plx1. Molecular and Cellular Biology 19: 8625-8632.

[0262] Ramalho-Santos, J., et al., Dev. Biol. 223, 54 (2000).

[0263] Resing, K. A., Mansour, S. J., Hermann, A. S., Johnson, R. S.,Candia, J. M., Fukasawa, K., Vande Woude, G. F., and Ahn, N. G. (1995).Determination of v-Mos-catalyzed phosphorylation sites andautophosphorylation sites on MAP kinase kinase by ESI/MS. Biochemistry34: 2610-2620.

[0264] Roberts, T. M. (1983). Crawling C. elegans spermatozoa contactthe substrate only by their pseudopods and contain 2-nm filaments. CellMotility 3: 333-347.

[0265] Roberts, T. M., Pavalko, F. M., and Ward, S. (1986). Membrane andcytoplasmic proteins are transported in the same organelle complexduring nematode spermatogenesis. Journal of Cell Biology 102: 1787-1796.

[0266] Roberts, T. M., and Stewart, M. (1995). Nematode spermlocomotion. Current Opinion in Cell Biology 7: 13-17.

[0267] Roberts, T. M., and Stewart, M. (2000). Acting like actin. Thedynamics of the nematode major sperm protein (msp) cytoskeleton indicatea push-pull mechanism for amoeboid cell motility. Journal of CellBiology 149: 7-12.

[0268] Roberts, T. M., and Ward, S. (1982a). Membrane flow duringnematode spermiogenesis. Journal of Cell Biology 92: 113-120.

[0269] Roberts, T. M., and Ward, S. (1982b). Centripetal flow ofpseudopodial surface components could propel the amoeboid movement of C.elegans spermatozoa. Journal of Cell Biology 92: 132-138.

[0270] Rose, K. L., Winfrey, V. P., Hoffman, L. H., Hall, D. H., Furuta,T., and Greenstein D. (1997). The POU gene ceh-18 promotes gonadalsheath cell differentiation and function required for meiotic maturationand ovulation in Caenorhabditis elegans. Developmental Biology 192:59-77.

[0271] Rutledge, E., Bianchi, L., Christensen, M., Morrison, R.,Broslat, A., Beld, A., George Jr., A. L., Greenstein, D., and Strange,K. (2000). CLH-3: a C. elegans ClC-2 chloride channel orthologueregulated by cell cycle events and involved in soma-germlineintercellular signaling. Submitted.

[0272] Sagata, N. (1997). What does Mos do in oocytes and somatic cells?BioEssays 19: 13-21.

[0273] Sagata, N., Oskarsson, M., Copeland, T., Brumbaugh, J., and VandeWoude, G. F. (1988). Function of c-mos proto-oncogene product in meioticmaturation in Xenopus oocytes. Nature 335:519-525.

[0274] Sagata, N., Watanabe, N., Vande Woude, G. F., and Ikawa, Y.(1989). The c-mos proto-oncogene product is a cytostatic factorresponsible for meiotic arrest in vertebrate eggs. Nature 342: 512-518.

[0275] Schagger, H., and von Jagow, G. (1987). Tricine-sodium dodecylsulfate-polyacrylamide gel electrophoresis for the separation ofproteins in the range from 1 to 100 kDa. Analytical Biochemistry 166:368-379.

[0276] Schedl, T., and Kimble, J. (1988). fog-2, a germ-line-specificsex determination gene required for hermaphrodite spermatogenesis in C.elegans. Genetics 119: 43-61.

[0277] Schulz, J. R., et al., J. Biol. Chem. 273, 24355 (1998).

[0278] Seed, B., and Aruffo, A. (1987). Molecular cloning of the CD2antigen, the T-cell erythrocyte receptor, by a rapid immunoselectionprocedure. Proceedings of the National Academy of Sciences USA 84:3365-3369.

[0279] Shakes, D. C., and Ward, S. (1989). Initiation of spermiogenesisin C. elegans: A pharmacological and genetic analysis. DevelopmentalBiology 134: 189-200.

[0280] Sheets, M. D., Wu, M., and Wickens, M. (1995). Polyadenylation ofc-mos mRNA as a control point in Xenopus meiotic maturation. Nature 374:511-516.

[0281] Skehel, P. A., Armitage, B. A., Bartsch, D., Hu, Y., Kaang, B.K., Siegelbaum, S. A., Kandel, E. R., and Martin, K. C. (1995). Proteinsfunctioning in synaptic transmission at the sensory to motor synapse ofAplysia. Neuropharmacology 34: 1379-1385.

[0282] Skehel, P. A. et al., Science 269, 1580 (1995).

[0283] Smith, H. E., and Ward, S. (1998). Identification ofprotein-protein interactions of the major sperm protein (MSP) ofCaenorhabditis elegans. Journal of Molecular Biology 279: 605-619.

[0284] Smith, L. D., and Ecker, R. E. (1969). Role of the oocyte nucleusin physiological maturation in Rana pipiens. Developmental Biology 19:281-309.

[0285] Smith, L. D., and Ecker, R. E. (1971). The interaction ofsteroids with Rana pipiens oocytes in the induction of maturation.Developmental Biology 25: 232-247.

[0286] Soussan, L. et al., J. Cell Biol. 146, 301 (1999).

[0287] Spector, D. L., Goldman, R. D., and Leinwand, L. A. (1998).Plasma membrane isolation using the cationic colloidal silica isolationtechnique. In Culture and Biochemical Analysis of Cells. K. Janssen,editor. p. 35.1-35.14. Cold Spring Harbor Laboratory Press: Plainview,N.Y.

[0288] Starck, J., Gibert, M. -A., Brun, J., and Bosch C. (1983),Ribosomal RNA synthesis and processing during oogenesis of the freeliving nematode C. elegans. Comparative Biochemistry and Physiology 75B:575-580.

[0289] Stebbins-Boaz, B., Hake, L. E., and Richter, J. D. (1996). CPEBcontrols the cytoplasmic polyadenylation of cyclin, Cdk2 and c-mos mRNAsand is necessary for oocyte maturation in Xenopus. EMBO Journal 5:2582-2592.

[0290] Strome, S. (1986). Fluorescence visualization of the distributionof microfilaments in gonads and early embryos of the nematode C.elegans. Journal of Cell Biology 103: 2241-2252.

[0291] Sulston, J., and Hodgkin, J. (1988). Methods. In The NematodeCaenorhabditis elegans. W. B. Wood, editor, p. 587-606. Cold SpringHarbor Laboratory Press, Cold Spring Harbor, N.Y.

[0292] Swofford, D., “PAUP: Phylogenetic analysis using parsimony.”Illinois National History Survey, Champaign (1993).

[0293] Vacquier, V. D., Science 281, 1995 (1998).

[0294] Veenstra, J. A. (2000). Mono- and dibasic proteolytic cleavagesites in insect neuroendocrine peptide precursors. Archives of InsectBiochemistry and Physiology 43: 49-63.

[0295] Ward, S. (1986). Asymmetric localization of gene products duringthe development of C. elegans spermatozoa. In Gametogenesis and theEarly Embryo. 44th Symposium of the Society for Developmental Biology.Gall, J. G., (ed). p.55-75. Alan R. Liss, NY.

[0296] Ward, S., Argon, Y., and Nelson, G. A. (1981). Spermmorphogenesis in wild-type and fertilization-defective mutants of C.elegans. Journal of Cell Biology 91: 26-44.

[0297] Ward, S., Burke, D. J., Sulston, J. E., Coulson, A. R.,Albertson, D. G., Ammons, D., Klass, M., and Hogan, E. (1988). Genomicorganization of major sperm protein genes and pseudogenes in thenematode Caenorhabditis elegans. Journal of Molecular Biology 199, 1-13.

[0298] Ward, S., and Carrel, J. S. (1979). Fertilization and spermcompetition in the nematode Caenorhabditis elegans. DevelopmentalBiology 73: 304-321.

[0299] Ward, S., Hogan, E., and Nelson, G. A. (1983). The initiation ofspermiogenesis in the nematode C. elegans. Developmental Biology 98:70-79.

[0300] Ward, S., and Klass, M. (1982). The location of the major proteinin C. elegans sperm and spermatocytes. Developmental Biology 92:203-208.

[0301] Ward, S., and Miwa, J. (1978). Characterization oftemperature-sensitive, fertilization-defective mutants of the nematodeC. elegans. Genetics 88: 285-303.

[0302] Ward, S., Roberts, T. M., Strome, S., Pavalko, F. M., and Hogan,E. (1986). Monoclonal antibodies that recognize a polypeptide antigenicdeterminant shared by multiple C. elegans sperm-specific proteins.Journal of Cell Biology 102: 1778-1786.

[0303] Wimalawansa, S. J. (1995). Purification and biochemicalcharacterization of Neuropeptide Y2 receptor. The Journal of BiologicalChemistry 270: 18523-18530.

[0304] Wolf, N., Hirsh, D., and McIntosh, J. R. (1978). Spermatogenesisin males of the free-living nematode, C. elegans. Journal ofUltrastructure Research 63: 155-169.

[0305] Yasunaga, S., Grati, M., Cohen-Salmon, M., El-Amraoui, A.,Mustapha, M., Salem, N., El-Zir, E., Loiselet, J., and Petit, C. (1999).A mutation in OTOF, encoding otoferlin, a FER-1-like protein, causesDFNB9, a nonsyndromic form of deafness. Nature Genetics 21:363-369.

[0306] Yew, N., Mellini, M. L., and Vande Woude, G. F. (1992). Meioticinitiation by the mos protein in Xenopus. Nature 355: 649-652.

[0307] Yung, Y., et al., FEBS Lett. 408, 292 (1997).

[0308] 4. To prepare SCM, purified sperm (26) were incubated in M9buffer (˜5×10⁷ sperm/ml) for 1-16 hrs at 20° C. Sperm were removed bycentrifugation for 5 min at 14,000 rpm in an Eppendorf microcentrifuge(model 5415C) and filtration through a 0.22 μm cellulose acetate filter(Costar). Samples (˜50 pl) were microinjected into the uterus offog-2(q71) (3) adult females (30 hrs post-L4 at 20° C.). Followinginjection, females were anesthetized for 20 minutes with 0.1%tricaine/0.01% tetramisole (32) in M9. Oocyte maturation and sheath cellcontraction rates were monitored by time-lapse video microscopy (1) for70 minutes.

[0309] 5. SCM or sperm lysates (by vortexing with glass beads) werefractionated on C₄ and C₁₈ columns (Vydac) using an acetonitrilegradient (0-100%) mobile phase. TFA (0.1%) was added to the mobile phaseto sharpen peaks by ion pairing. Absorbance peaks (214 nm) werecollected manually, dialyzed against M9, and bioassayed. Activefractions were analyzed by MALDI-TOF mass spectrometry using internalmolecular weight standards (insulin, cytochrome C and myoglobin).

[0310] 6. Post source decay mass spectrometry (33) of a 1960 Da peptide,generated by tryptic digestion of the active fraction, yielded thesequence IVFNAPYDDKHTYHIK, which matched MSP.

[0311] 9. His-tagged MSP-77, MSP-38, and MSP(1-106) were expressed inEscherichia coli M15[pRep4] (Qiagen) and purified under nativeconditions by Ni-NTA affinity chromatography (>99% pure by SDS-PAGE andmass spectrometry). The extinction coefficient c (275 nm) of MSP wasestimated by amino acid analysis of a purified His-tagged MSP-77standard. MSP concentrations were determined by amino acid hydrolysis,SDS-PAGE, and spectrophotometrically using ε (275 nm)=3.29×10⁴M⁻¹cm⁻¹.

[0312] 10. Anti-MSP (26) or control EMB-30 antibodies (34) were injected(˜40 μg/ml) into adult wild-type N2 hermaphrodites (24 hr post-L4 at 20°C.). The injected animals were cultured individually with food for a 3hr time period and total ovulations were determined. The effect ofantibody injection on oocyte maturation was determined by time lapsevideo microscopy. A two-sample t-test was used to compare results of MSPinjections with controls.

[0313] 12. The C-terminal MSP peptide (EWFQGDGMVRRKNLPIEYNP) wasprepared by solid-phase synthesis and purified by HPLC (ResearchGenetics).

[0314] 15. Diphosphorylated MAP kinase was detected in dissected andfixed (3% paraformaldehyde) gonadal preparations using indirectimmunofluorescence with the antibody MAPK-YT (35) (Sigma). In C. eleganspreparations, MAPK-YT only recognizes mpk-1 map kinase gene products(36). Gonads were stained 8, 40, or 50 min post MSP injection. ActivatedMAP kinase was detected 40 and 50 min post-injection but was notdetectable 8 min post injection.

[0315] 22. Phylogenetic analyses were performed using maximum parsimonyand neighbor-joining methods. Amino acid sequences from MSP and MSP-likedomains of several representative VAPs were used in the analyses. Forparsimony, the heuristic search option of PAUP* 3.1 (37) was used fortree construction, with 200 random order taxon addition replicates andtree bisection and reconnection branch swapping. Bacterial PapD, whichis structurally related to MSP, was used as the outgroup. The “protpars”matrix of PAUP* 3.1 was used to weigh amino acid substitutions. Toobtain bootstrap values, 100 bootstrap replicates were performed usingsimple taxon addition with tree bisection and reconnection branchswapping.

[0316] The present invention is not limited by mechanism or theory.Although there have been described general and specific embodiments ofthe invention herein, these embodiments do not limit the scope of theinvention except as set forth in the claims below.

What is claimed is:
 1. A method of identifying an anti-nematode agent,comprising: contacting a test compound to a nematode; and monitoring afemale sexual maturation of the nematode, wherein inhibition of thefemale sexual maturation indicates that the test compound includes theanti-nematode agent.
 2. The method of claim 1, wherein the female sexualmaturation comprises an oocyte maturation or a gonadal sheath cellcontraction.
 3. The method of claim 1, wherein the female sexualmaturation comprises an ovulation.
 4. The method of claim 1, wherein themonitoring step includes optical detection.
 5. The method of claim 1,wherein the monitoring step includes optical detection of more than onefemale sexual maturation episode at the same time.
 6. The method ofclaim 1, wherein the nematode is an animal parasitic nematode.
 7. Themethod of claim 1, wherein the nematode is a plant parasitic nematode.8. The method of claim 1, wherein the nematode is of the genusCaenorhabditis.
 9. The method of claim 1, wherein the nematode isCaenorhabditis elegans.
 10. The method of claim 1, wherein the nematodeis of the genus Ascaris.
 11. The method of claim 1, wherein the nematodecomprises a roundworm.
 12. The method of claim 1, wherein the nematodecomprises a member of a genus selected from a group consisting of:Ascaris, Heterodera, Globodera, Meloidogyne, Ditylenchus, Anguina,Pratylenchus, Radopholus, Hirschmanniella, Hoplolaimus, Rotylenchulus,Tylenchulus, Helicotylenchus, Criconemella, Xiphinema, Longidorus,Trichodorus, Paratrichodorus, Aphelenchs, Onchocerca, Brugia, andWuchereria.
 13. The method of claim 1, wherein the nematode includes adefect in a production of a sperm.
 14. The method of claim 1, whereinthe nematode includes a genetic mutation.
 15. The method of claim 1,wherein the nematode is selected from a group consisting of: a fog-1nematode, a fog-2 nematode, a fog-3 nematode, a fem-1 nematode, a fem-2nematode, a fem-3 nematode, and a gld-1 nematode.
 16. The method ofclaim 1, wherein the nematode comprises a transgenic nematode includinga transgenic expression of a major sperm protein, or a biologicallyactive fragment thereof.
 17. The method of claim 1, wherein the nematodecomprises a transgenic nematode including an ectopic expression of amajor sperm protein, or a biologically active fragment thereof.
 18. Themethod of claim 1, wherein the nematode comprises a transgenic nematodeincluding a transgenic expression of a major sperm protein from anematode of a genus Caenorhabditis or a genus Ascaris, or a biologicallyactive fragment thereof.
 19. A method of inhibiting a reproduction ofthe nematode of claim 1, comprising: administering the anti-nematodeagent identified in claim 1 to the nematode.
 20. The method of claim 19,wherein the anti-nematode agent comprises an antibody that bindsspecifically to the major sperm protein, or a biologically activefragment thereof.
 21. A method of controlling a population of nematodes,comprising: administering the anti-nematode agent identified in claim 1to the nematodes.
 22. A method of controlling a population of nematodesin an animal in need thereof, comprising: administering theanti-nematode agent identified in claim 1 to the animal in an amounteffective to control the population of nematodes.
 23. A method ofincreasing a host resistance to a nematode infection in an animal inneed thereof, comprising: administering the anti-nematode agentidentified in claim 1 to the animal in an amount effective to increasethe host resistance.
 24. A method of increasing a host resistance to anematode infection in a non-human animal in need thereof, comprising:expressing the anti-nematode agent identified in claim 1 in the animal,wherein the agent comprises a polypeptide which binds a major spermprotein.
 25. A method of controlling a population of nematodes in aplant in need thereof, comprising: administering the anti-nematode agentidentified in claim 1 to the plant in an amount effective to control thepopulation of nematodes.
 26. A method of increasing a host resistance toa nematode infection in a plant in need thereof, comprising:administering the anti-nematode agent identified in claim 1 to the plantin an amount effective to increase the host resistance.
 27. A method ofincreasing a host resistance to a nematode infection in an plant in needthereof, comprising: expressing the anti-nematode agent identified inclaim 1 in the plant, wherein the agent comprises a polypeptide whichbinds a major sperm protein.
 28. A process of making a factor thatinhibits a female sexual maturation of a nematode, the methodcomprising: contacting a test compound to a nematode; monitoring thefemale sexual maturation, wherein inhibition of the female sexualmaturation indicates that the test compound comprises the factor; andmanufacturing the factor.
 29. A method of identifying an anti-nematodeagent, comprising: contacting a test compound to a polypeptidecomprising a major sperm protein or a portion thereof capable ofstimulating a female sexual maturation; and detecting a compositionincluding the test compound and the polypeptide, wherein the presence ofthe composition indicates that the test compound is the anti-nematodeagent.
 30. The method of claim 29, further comprising screening theanti-nematode agent for inhibition of a female sexual maturation. 31.The method of claim 29, wherein the MSP comprises the amino acidsequence set forth in SEQ ID NO:2: or nematode major sperm proteinalignment variants thereof, or a portion thereof capable of stimulatinga female sexual maturation.
 32. A method of inhibiting a reproduction ofa nematode, comprising: inhibiting a stimulation of a femalereproductive cell by a major sperm protein.
 33. A method of identifyingan agent that binds to a biologically active domain of a nematode majorsperm protein, comprising: contacting a test compound to the domain; anddetecting a composition including the test compound and the domain,wherein the presence of the composition indicates that the agent bindsto the domain.
 34. An isolated nematode major sperm protein domain,comprising: a polypeptide including 125 or fewer consecutive amino acidsof a nematode major sperm protein, wherein the domain is capable ofstimulating a female sexual maturation.
 35. The major sperm proteindomain of claim 34, wherein the polypeptide includes 20 or fewer aminoacids of the nematode major sperm protein, wherein the domain is capableof stimulating the female sexual maturation.
 36. The major sperm proteindomain of claim 34, wherein the polypeptide includes 10 to 30 aminoacids of the nematode major sperm protein, wherein the domain is capableof stimulating the female sexual maturation.
 37. An isolated nematodemajor sperm protein domain, comprising: a polypeptide including 125 orfewer consecutive amino acids as set forth in SEQ ID NO:2 or nematodemajor sperm protein alignment variants thereof, wherein the domain iscapable of stimulating a female sexual maturation.
 38. The major spermprotein domain of claim 37, wherein the polypeptide includes 20 or feweramino acids, wherein the domain is capable of stimulating a femalesexual maturation.
 39. The major sperm protein domain of claim 37,wherein the polypeptide includes 10 to 30 amino acids, wherein thedomain is capable of stimulating a female sexual maturation.
 40. Anantibody that selectively binds to a biologically active domain of anematode major sperm protein, wherein the biologically active domaincomprises a sheath cell contraction domain.
 41. The antibody of claim40, wherein the biologically active domain comprises 20 or fewer aminoacids of a carboxyl-terminus of the domain.
 42. An antibody thatselectively binds to a biologically active domain of a nematode majorsperm protein, wherein the biologically active domain comprises anoocyte maturation domain.
 43. The antibody of claim 42, wherein thebiologically active domain includes an amino acid sequence having agroup of consecutive residues in the range of 96-110 of SEQ ID NO:2, ornematode major sperm protein alignment variants thereof; and wherein theamino acid sequence includes 20 or fewer consecutive residues of SEQ IDNO:2, or nematode major sperm protein alignment variants thereof. 44.The antibody of claim 42, wherein the biologically active domainincludes an amino acid sequence having a group of consecutive residuesin the range of 1-106 of SEQ ID NO:2, or nematode major sperm proteinalignment variants thereof; and wherein the amino acid sequence includes20 or fewer consecutive residues of SEQ ID NO:2, or nematode major spermprotein alignment variants thereof.
 45. A process of making an antibody,comprising immunizing a non-human animal with an immunogenic fragment ofa nematode major sperm protein domain including a polypeptide having 20or fewer consecutive amino acids as set forth in SEQ ID NO:2 or nematodemajor sperm protein alignment variants thereof, wherein the domain iscapable of stimulating a female sexual maturation.